Antisense oligonucleotide compositions and methods for the modulation of JNK proteins

ABSTRACT

Compositions and methods for the treatment and diagnosis of diseases or disorders amenable to treatment through modulation of expression of a gene encoding a Jun N-terminal kinase (JNK protein) are provided. Oligonucleotide are herein provided which are specifically hybridizable with nucleic acids encoding JNK1, JNK2 and JNK3, as well as other JNK proteins and specific isoforms thereof. Methods of treating animals suffering from diseases or disorders amenable to therapeutic intervention by modulating the expression of one or more JNK proteins with such oligonucleotide are also provided. Methods for the treatment and diagnosis of diseases or disorders associated with aberrant expression of one or more JNK proteins are also provided. Methods for inducing apoptosis and for treating diseases or conditions associated with a reduction in apoptosis are also provided.

INTRODUCTION

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/396,902 filed on Sep. 15, 1999, which is acontinuation-in-part of U.S. application Ser. No. 09/287,796, filed onApr. 7, 1999, now issued U.S. Pat. No. 6,133,246, which is acontinuation-in-part of U.S. application Ser. No. 09/130,616 filed onAug. 7, 1998 which is a continuation-in-part of U.S. application Ser.No. 08/910,629 filed on Aug. 13, 1997, now issued U.S. Pat. No.5,877,309.

FIELD OF THE INVENTION

[0002] The present invention provides compositions and methods fordetecting and modulating levels of Jun N-terminal kinases (JNKproteins), enzymes which are encoded by JNK genes. In particular, theinvention relates to antisense oligonucleotides specificallyhybridizable with nucleic acids encoding JNK proteins. It has been foundthat antisense oligonucleotides can modulate the expression of these andother JNK proteins, kinases which were initially discovered due to theirability to catalyze the phosphorylation of the c-Jun subunit oftranscription factor AP-1 and thereby increase AP-1 activity. Othertranscription factors, such as AT-2, are similarly activated by JNKproteins, and a variety of other cellular effectors may serve assubstrates for JNK proteins (Gutta et al., Science, 1995, 267, 389). Inany event, transcription factor AP-1 has been implicated in abnormalcell proliferation, oncogenic transformation, and tumor formation,development and maintenance (Volt, Chapter 15 In: The FOS and JUNFamilies of Transcription Factors, Angel and Herrlich, Eds., CBC Press,Boca Raton, Fla., 1994). Accordingly, it is believed that (1) JNKproteins are aberrantly expressed in some neoplasms and tumors withresultant increased AP-1 activity, and (2) even in abnormallyproliferating cells in which a JNK gene is not aberrantly expressed,inhibition of JNK expression will result in decreased AP-1 activity andthus, inhibition of abnormal cell proliferation and tumor formation,development and maintenance. The invention is thus directed todiagnostic methods for detecting, and therapeutic methods forinhibiting, the hyperproliferation of cells and the formation,development and maintenance of tumors. Furthermore, this invention isdirected to treatment of conditions associated with abnormal expressionof JNK genes. This invention also relates to therapies, diagnostics, andresearch reagents for disease states or disorders which respond tomodulation of the expression of JNK proteins. Inhibition of thehyperproliferation of cells, and corresponding prophylactic, palliativeand therapeutic effects result from treatment with the oligonucleotidesof the invention.

BACKGROUND OF THE INVENTION

[0003] Transcription factors play a central role in the expression ofspecific genes upon stimulation by extracellular signals, therebyregulating a complex array of biological processes. Members of thefamily of transcription factors termed AP-1 (activating protein-1) altergene expression in response to growth factors, cytokines, tumorpromoters, carcinogens and increased expression of certain oncogenes(Rahmsdorf, Chapter 13, and Rapp et al., Chapter 16 In: The FOS and JUNFamilies of Transcription Factors, Angel and Herrlich, Eds., CBC Press,Boca Raton, Fla., 1994). Growth factors and cytokines, such as TNFa,exert their function by binding to specific cell surface receptors.Receptor occupancy triggers a signal transduction cascade to thenucleus. In this cascade, transcription factors such as AP-1 executelong term responses to the extracellular factors by modulating geneexpression. Such changes in cellular gene expression lead to DNAsynthesis, and eventually the formation of differentiated derivatives(Angel and Karin, Biochim. Biophys. Acta, 1991, 1072, 129).

[0004] In general terms, AP-1 denotes one member of a family of relatedheterodimeric transcription factor complexes found in eukaryotic cellsor viruses (The FOR and JUN Families of Transcription Factors, Angel andHairlike, Eds., CBC Press, Boca Raton, Fla., 1994; Bohmann et al.,Science, 1987, 238, 1386; Angel et al., Nature, 1988, 332, 166). Tworelatively well-characterized AP-1 subunits are c-For and c-Jun; thesetwo proteins are products of the c-for and c-jun proto-oncogenes,respectively. Repression of the activity of either c-for or c-jun, or ofboth proto-oncogenes, and the resultant inhibition of the formation ofc-For and c-Jun proteins, is desirable for the inhibition of cellproliferation, tumor formation and tumor growth.

[0005] The phosphorylation of proteins plays a key role in thetransduction of extracellular signals into the cell. Mitogen-activatedprotein kinases (MAPKs), enzymes which effect such phosphorylations aretargets for the action of growth factors, hormones, and other agentsinvolved in cellular metabolism, proliferation and differentiation (Cobbet al., J. Biol. Chem., 1995, 270, 14843). MAPKs (also referred to asextracellular signal-regulated protein kinases, or ERKs) are themselvesactivated by phosphorylation catalyzed by, e.g., receptor tyrosinekinases, G protein-coupled receptors, protein kinase C (PKC), and theapparently MAPK-dedicated kinases MEK1 and MEK2. In general, MAP kinasesare involved in a variety of signal transduction pathways (sometimesoverlapping and sometimes parallel) that function to conveyextracellular stimuli to protooncogene products to modulate cellularproliferation and/or differentiation (Seger et al., FASEB J., 1995, 9,726; Cano et al., Trends Biochem. Sci., 1995, 20, 117). In a typical MAPkinase pathway, it is thought that a first MAP kinase, called a MEKK,phosphorylates and thereby activates a second MAP kinase, called a MEK,which, in turn, phosphorylates and activates a MAPK/ERK or JNK/SAPKenzyme (“SAPK” is an abbreviation for stress-activated protein kinase).Finally, the activated MAPK/ERK or JNK/SAPK enzyme itself phosphorylatesand activates a transcription factor (such as, e.g., AP-1) or othersubstrates (Cano et al., Trends Biochem. Sci., 1995, 20, 117). Thiscanonical cascade can be simply represented as follows:

[0006] MEKK→MEK→MAPK/ERK→transcription factor or JNK/SAPK or othersubstrate(s)

[0007] One of the signal transduction pathways involves the MAP kinasesJun N-terminal kinase 1 (JNK1) and Jun N-terminal kinase 2 (JNK2) whichare responsible for the phosphorylation of specific sites (Serine 63 andSerine 73) on the amino terminal portion of c-Jun. Phosphorylation ofthese sites potentiates the ability of AP-1 to activate transcription(Binetruy et al., Nature, 1991, 351, 122; Smeal et al., Nature, 1991,354, 494). Besides JNK1 and JNK2, other JNK family members have beendescribed, including JNK3 (Gutta et al., EMBO J., 1996, 15, 2760),initially named p49^(3F12) kinase (Mohit et al., Neuron, 1994, 14, 67).The term “JNK protein” as used herein shall mean a member of the JNKfamily of kinases, including but not limited to JNK1, JNK2 and JNK3,their isoforms (Gutta et al., EMBO J., 1996, 15, 2760) and other membersof the JNK family of proteins whether they function as Jun N-terminalkinases per se (that is, phosphorylate Jun at a specific aminoterminally located position) or not.

[0008] At least one human leukemia oncogene has been shown to enhanceJun N-terminal kinase function (Raitano et al., Proc. Natl. Acad. Sci.(USA), 1995, 92, 11746). Modulation of the expression of one or more JNKproteins is desirable in order to interfere with hyperproliferation ofcells and with the transcription of genes stimulated by AP-1 and otherJNK protein phosphorylation substrates. Modulation of the expression ofone or more other JNK proteins is also desirable in order to interferewith hyperproliferation of cells resulting from abnormalities inspecific signal transduction pathways. To date, there are no knowntherapeutic agents which effectively inhibit gene expression of one ormore JNK proteins. Consequently, there remains a long-felt need forimproved compositions and methods for modulating the expression ofspecific JNK proteins.

[0009] Moreover, cellular hyperproliferation in an animal can haveseveral outcomes. Internal processes may eliminate hyperproliferativecells before a tumor can form. Tumors are abnormal growths resultingfrom the hyperproliferation of cells. Cells that proliferate to excessbut stay put form benign tumors, which can typically be removed by localsurgery. In contrast, malignant tumors or cancers comprise cells thatare capable of undergoing metastasis, i.e., a process by whichhyperproliferative cells spread to, and secure themselves within, otherparts of the body via the circulatory or lymphatic system (see,generally, Chapter 16 In: Molecular Biology of the Cell, Alberts et al.,Eds., Garland Publishing, Inc., New York, 1983). Using antisenseoligonucleotides, it has surprisingly been discovered that several genesencoding enzymes required for metastasis are positively regulated byAP-1, which may itself be modulated by antisense oligonucleotidestargeted to one or more JNK proteins. Consequently, the inventionsatisfies the long-felt need for improved compositions and methods formodulating the metastasis of malignant tumors.

[0010] Prostate cancer is the most commonly diagnosed malignancy inAmerican men. Therapy for advanced prostate cancer generally involvescastration or drug therapy to remove or suppress androgens. Progressionto androgen-independence inevitably occurs, associated with thedevelopment of clinical symptoms, particularly metastases to the bone,and rising serum prostate specific antigen levels. Conventionalcytotoxic chemotherapy is generally ineffective (response rate below10%) or poorly tolerated in the elderly male population.

[0011] c-jun has been shown to selectively activate androgenreceptor-dependent transactivation. Consequently, c-jun has beenimplicated as a possible mediator of prostate tumor progression afterandrogen withdrawal, thus c-jun and the JNK pathway are potentialchemotherapeutic targets. Bubulya et al., J. Biol. Chem. 1996, 271,24583-24589.

[0012] JNKs have been implicated as key mediators of a variety ofcellular responses and pathologies. JNKs can be activated byenvironmental stress, such as radiation, heat shock, osmotic shock, orgrowth factor withdrawal as well as by pro-inflammatory cytokines.Several studies have demonstrated a role for JNK activation in apoptosisinduced by a number of stimuli in several cell types. Apoptosis, orprogrammed cell death, is an essential feature of growth anddevelopment, as the control of cell number is a balance between cellproliferation and cell death. Apoptosis is an active rather than apassive process, resulting in cell suicide as a result of any of anumber of external or internal signals. Apoptotic cell death ischaracterized by nuclear condensation, endonucleolytic degradation ofDNA at nucleosomal intervals (“laddering”) and plasma membrane blebbing.Programmed cell death plays an essential role in, for example, immunesystem development and nervous system development. In the former, Tcells displaying autoreactive antigen receptors are removed byapoptosis. In the latter, a significant reshaping of neural structuresoccurs, partly through apoptosis.

[0013] Diseases and conditions in which apoptosis has been implicatedfall into two categories, those in which there is increased cellsurvival (i.e., apoptosis is reduced) and those in which there is excesscell death (i.e.,apoptosis is increased). Diseases in which there is anexcessive accumulation of cells due to increased cell survival includecancer, autoimmune disorders and viral infections. Until recently, itwas thought that cytotoxic drugs killed target cells directly byinterfering with some life-maintaining function. However, of late, ithas been shown that exposure to several cytotoxic drugs with disparatemechanisms of action induces apoptosis in both malignant and normalcells. Manipulation of levels of trophic factors (e.g., by anti-estrogencompounds or those which reduce levels of various growth hormones) hasbeen one clinical approach to promote apoptosis, since deprivation oftrophic factors can induce apoptosis. Apoptosis is also essential forthe removal of potentially autoreactive lymphocytes during developmentand the removal of excess cells after the completion of an immune orinflammatory response. Recent work has clearly demonstrated thatimproper apoptosis may underlie the pathogenesis of autoimmune diseasesby allowing abnormal autoreactive lymphocytes to survive. For these andother conditions in which insufficient apoptosis is believed to beinvolved, promotion of apoptosis is desired.

[0014] In the second category, AIDS and neurodegenerative disorders likeAlzheimer's or Parkinson's disease represent disorders for which anexcess of cell death due to promotion of apoptosis (or unwantedapoptosis) has been implicated. Amyotrophic lateral sclerosis, retinitispigmentosa, and epilepsy are other neurologic disorders in whichapoptosis has been implicated. Apoptosis has been reported to occur inconditions characterized by ischemia, e.g. myocardial infarction andstroke. Apoptosis has also been implicated in a number of liverdisorders including obstructive jaundice and hepatic damage due totoxins and drugs. Apoptosis has also been identified as a key phenomenonin some diseases of the kidney, i.e. polycystic kidney, as well as indisorders of the pancreas including diabetes. Thatte, U. et al., 1997,Drugs 54, 511-532. For these and other diseases and conditions in whichunwanted apoptosis is believed to be involved, inhibitors of apoptosisare desired.

SUMMARY OF THE INVENTION

[0015] In accordance with the present invention, oligonucleotides areprovided which specifically hybridize with a nucleic acid encoding a JNKprotein. Certain oligonucleotides of the invention are designed to bindeither directly to mRNA transcribed from, or to a selected DNA portionof, a JNK gene that encodes a JNK protein, thereby modulating theexpression thereof and/or the phosphorylation of one or more substratesfor the JNK protein. Pharmaceutical compositions comprising theoligonucleotides of the invention, and various methods of using theoligonucleotides of the invention, including methods of modulating oneor more metastatic events, are also herein provided.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides are commonly described as“antisense.” Antisense oligonucleotides are commonly used as researchreagents, diagnostic aids, and therapeutic agents. It has beendiscovered that genes (JNK) encoding Jun N-terminal kinase (JNKproteins) are particularly amenable to this approach. In the context ofthe invention, the terms “Jun N-terminal kinase” and “JNK protein” referto proteins actually known to phosphorylate the amino terminal(N-terminal) portion of the Jun subunit of AP-1, as well as those thathave been tentatively identified as JNK proteins based on amino acidsequence but which may in fact additionally or alternatively bind and/orphosphorylate either other transcription factors (e.g., ATF2) or kinasesubstrates that are not known to be involved in transcription (Derijardet al., Cell, 1994, 76, 1025; Kallunki et al., Genes & Development,1994, 8, 2996; Gutta et al., EMBO J., 1996, 15, 2760). Morespecifically, the present invention is directed to antisenseoligonucleotides that modulate the JNK1, JNK2 and JNK3 proteins. As aconsequence of the association between cellular proliferation andactivation (via phosphorylation) of AP-1, other transcription factorsand/or other proteins by JNK proteins, inhibition of the expression ofone or more JNK proteins leads to inhibition of the activation of AP-1and/or other factors involved in cellular proliferation, cell cycleprogression or metastatic events, and, accordingly, results inmodulation of these activities. Such modulation is desirable fortreating, alleviating or preventing various hyperproliferative disordersor diseases, such as various cancers. Such inhibition is furtherdesirable for preventing or modulating the development of such diseasesor disorders in an animal suspected of being, or known to be, prone tosuch diseases or disorders. If desired, modulation of the expression ofone JNK protein can be combined with modulation of one or moreadditional JNK proteins in order to achieve a requisite level ofinterference with AP-1-mediated transcription.

[0017] Methods of modulating the expression of JNK proteins comprisingcontacting animals with oligonucleotides specifically hybridizable witha nucleic acid encoding a JNK protein are herein provided. These methodsare believed to be useful both therapeutically and diagnostically as aconsequence of the association between kinase-mediated activation ofAP-1 and cellular proliferation. These methods are also useful as tools,for example, in the detection and determination of the role ofkinase-mediated activation of AP-1 in various cell functions andphysiological processes and conditions, and for the diagnosis ofconditions associated with such expression and activation.

[0018] The present invention also comprises methods of inhibitingJNK-mediated activation using the oligonucleotides of the invention.Methods of treating conditions in which abnormal or excessiveJNK-mediated cellular proliferation occurs are also provided. Thesemethods employ the oligonucleotides of the invention and are believed tobe useful both therapeutically and as clinical research and diagnostictools. The oligonucleotides of the present invention may also be usedfor research purposes. Thus, the specific hybridization exhibited by theoligonucleotides of the present invention may be used for assays,purifications, cellular product preparations and in other methodologieswhich may be appreciated by persons of ordinary skill in the art.

[0019] The present invention employs oligonucleotides for use inantisense modulation of the function of DNA or messenger RNA (mRNA)encoding a protein the modulation of which is desired, and ultimately toregulate the amount of such a protein. Hybridization of an antisenseoligonucleotide with its mRNA target interferes with the normal role ofmRNA and causes a modulation of its function in cells. The functions ofmRNA to be interfered with include all vital functions such astranslocation of the RNA to the site for protein translation, actualtranslation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and possibly even independent catalytic activitywhich may be engaged in by the RNA. The overall effect of suchinterference with mRNA function is modulation of the expression of aprotein, wherein “modulation” means either an increase (stimulation) ora decrease (inhibition) in the expression of the protein. In the contextof the present invention, inhibition is the preferred form of modulationof gene expression.

[0020] It is preferred to target specific genes for antisense attack.“Targeting” an oligonucleotide to the associated nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or MRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a foreign nucleic acid from aninfectious agent. In the present invention, the target is a cellulargene associated with hyperproliferative disorders. The targeting processalso includes determination of a site or sites within this gene for theoligonucleotide interaction to occur such that the desired effect,either detection or modulation of expression of the protein, willresult. Once the target site or sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity to give the desired effect. Generally, there are fiveregions of a gene that may be targeted for antisense modulation: the 5′untranslated region (hereinafter, the “5′-UTR”), the translationinitiation codon region (hereinafter, the “tIR”), the open reading frame(hereinafter, the “ORF”), the translation termination codon region(hereinafter, the “tTR”) and the 3′ untranslated region (hereinafter,the “3′-UTR”). As is known in the art, these regions are arranged in atypical messenger RNA molecule in the following order (left to right, 5′to 3′): 5′-UTR, tIR, ORF, tTR, 3′-UTR. As is known in the art, althoughsome eukaryotic transcripts are directly translated, many ORFs containone or more sequences, known as “introns,” which are excised from atranscript before it is translated; the expressed (unexcised) portionsof the ORF are referred to as “exons” (Alberts et al., Molecular Biologyof the Cell, 1983, Garland Publishing Inc., New York, pp. 411-415).Furthermore, because many eukaryotic ORFs are a thousand nucleotides ormore in length, it is often convenient to subdivide the ORF into, e.g.,the 5′ ORF region, the central ORF region, and the 3′ ORF region. Insome instances, an ORF contains one or more sites that may be targeteddue to some functional significance in vivo. Examples of the lattertypes of sites include intragenic stem-loop structures (see, e.g., U.S.Pat. No. 5,512,438) and, in unprocessed mRNA molecules, intron/exonsplice sites.

[0021] Within the context of the present invention, one preferredintragenic site is the region encompassing the translation initiationcodon of the open reading frame (ORF) of the gene. Because, as is knownin the art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon.” A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Furthermore, 5′-UUU functions as a translation initiation codon invitro (Brigstock et al., Growth Factors, 1990, 4, 45; Gelbert et al.,Somat. Cell. Mol. Genet., 1990, 16, 173; Gold and Stormo, in:Escherichia coli and Salmonella typhimurium: Cellular and MolecularBiology, Vol. 2, 1987, Neidhardt et al., Eds., American Society forMicrobiology, Washington, D.C., p. 1303). Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (prokaryotes). It is alsoknown in the art that eukaryotic and prokaryotic genes may have two ormore alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions, in order to generate relatedpolypeptides having different amino terminal sequences (Markussen etal., Development, 1995, 121, 3723; Gao et al., Cancer Res., 1995, 55,743; McDermott et al., Gene, 1992, 117, 193; Perri et al., J. Biol.Chem., 1991, 266, 12536; French et al., J. Virol., 1989, 63, 3270;Pushpa-Rekha et al., J. Biol. Chem., 1995, 270, 26993; Monaco et al., J.Biol. Chem., 1994, 269, 347; Devirgilio et al., Yeast, 1992, 8, 1043;Kanagasundaram et al., Biochim. Biophys. Acta, 1992, 1171, 198; Olsen etal., Mol. Endocrinol., 1991, 5, 1246; Saul et al., Appl. Environ.Microbiol., 1990, 56, 3117; Yaoita et al., Proc. Natl. Acad. Sci. USA,1990, 87, 7090; Rogers et al., EMBO J., 1990, 9, 2273). In the contextof the invention, “start codon” and “translation initiation codon” referto the codon or codons that are used in vivo to initiate translation ofan mRNA molecule transcribed from a gene encoding a JNK protein,regardless of the sequence(s) of such codons. It is also known in theart that a translation termination codon (or “stop codon”) of a gene mayhave one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (thecorresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively). The terms “start codon region” and “translationinitiation region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3′) from a translation initiation codon.Similarly, the terms “stop codon region” and “translation terminationregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′ or 3′) from a translation termination codon.

[0022] The remainder of the Detailed Description relates in more detailthe (1) Oligonucleotides of the Invention and their (2) Bioequivalents,(3) Utility, (4) Pharmaceutical Compositions and (5) Means ofAdministration.

[0023] 1. Oligonucleotides of the Invention

[0024] The present invention employs oligonucleotides for use inantisense modulation of one or more JNK proteins. In the context of thisinvention, the term “oligonucleotide” refers to an oligomer or polymerof ribonucleic acid or deoxyribonucleic acid. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent intersugar (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced binding to target and increased stability inthe presence of nucleases.

[0025] An oligonucleotide is a polymer of a repeating unit genericallyknown as a nucleotide. The oligonucleotides in accordance with thisinvention preferably comprise from about 8 to about 30 nucleotides. Anunmodified (naturally occurring) nucleotide has three components: (1) anitrogen-containing heterocyclic base linked by one of its nitrogenatoms to (2) a 5-pentofuranosyl sugar and (3) a phosphate esterified toone of the 5′ or 3′ carbon atoms of the sugar. When incorporated into anoligonucleotide chain, the phosphate of a first nucleotide is alsoesterified to an adjacent sugar of a second, adjacent nucleotide via a3′-5′ phosphate linkage. The “backbone” of an unmodified oligonucleotideconsists of (2) and (3), that is, sugars linked together byphosphodiester linkages between the 5′ carbon of the sugar of a firstnucleotide and the 3′ carbon of a second, adjacent nucleotide. A“nucleoside” is the combination of (1) a nucleobase and (2) a sugar inthe absence of (3) a phosphate moiety (Kornberg, A., DNA Replication, W.H. Freeman & Co., San Francisco, 1980, pages 4-7). The backbone of anoligonucleotide positions a series of bases in a specific order; thewritten representation of this series of bases, which is conventionallywritten in 5′ to 3′ order, is known as a nucleotide sequence.

[0026] Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides which specifically hybridize to aportion of the sense strand of a gene are commonly described as“antisense.” In the context of the invention, “hybridization” meanshydrogen bonding, which may be Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding, between complementary nucleotides. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementary orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. An oligonucleotide isspecifically hybridizable to its target sequence due to the formation ofbase pairs between specific partner nucleobases in the interior of anucleic acid duplex. Among the naturally occurring nucleobases, guanine(G) binds to cytosine (C), and adenine (A) binds to thymine (T) oruracil (U). In addition to the equivalency of U (RNA) and T (DNA) aspartners for A, other naturally occurring nucleobase equivalents areknown, including 5-methylcytosine, 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentiobiosyl HMC (C equivalents), and5-hydroxymethyluracil (U equivalent). Furthermore, synthetic nucleobaseswhich retain partner specificity are known in the art and include, forexample, 7-deaza-Guanine, which retains partner specificity for C. Thus,an oligonucleotide's capacity to specifically hybridize with its targetsequence will not be altered by any chemical modification to anucleobase in the nucleotide sequence of the oligonucleotide which doesnot significantly effect its specificity for the partner nucleobase inthe target oligonucleotide. It is understood in the art that anoligonucleotide need not be 100% complementary to its target DNAsequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when there is a sufficient degree ofcomplementary to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed.

[0027] Antisense oligonucleotides are commonly used as researchreagents, diagnostic aids, and therapeutic agents. For example,antisense oligonucleotides, which are able to inhibit gene expressionwith exquisite specificity, are often used by those of ordinary skill toelucidate the function of particular genes, for example to distinguishbetween the functions of various members of a biological pathway. Thisspecific inhibitory effect has, therefore, been harnessed by thoseskilled in the art for research uses. The specificity and sensitivity ofoligonucleotides is also harnessed by those of skill in the art fortherapeutic uses.

[0028] A. Modified Linkages

[0029] Specific examples of some preferred modified oligonucleotidesenvisioned for this invention include those containingphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are oligonucleotideswith phosphorothioates and those with CH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂[known as a methylene(methylimino) or MMI backbone], CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH₂). Also preferred areoligonucleotides having morpholino backbone structures (Summerton andWeller, U.S. Pat. No. 5,034,506). Further preferred are oligonucleotideswith NR—C(*)—CH₂—CH₂, CH₂—NR—C(*)—CH₂, CH₂—CH₂—NR—C(*), C(*)—NR—CH₂—CH₂and CH₂—C(*) —NR—CH₂ backbones, wherein “*” represents O or S (known asamide backbones; DeMesmaeker et al., WO 92/20823, published Nov. 26,1992). In other preferred embodiments, such as the peptide nucleic acid(PNA) backbone, the phosphodiester backbone of the oligonucleotide isreplaced with a polyamide backbone, the nucleobases being bound directlyor indirectly to the aza nitrogen atoms of the polyamide backbone(Nielsen et al., Science, 1991, 254, 1497; U.S. Pat. No. 5,539,082).

[0030] B. Modified Nucleobases

[0031] The oligonucleotides of the invention may additionally oralternatively include nucleobase modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include adenine (A),guanine (G), thymine (T), cytosine (C) and uracil (U). Modifiednucleobases include nucleobases found only infrequently or transientlyin natural nucleic acids, e.g., hypoxanthine, 6-methyladenine,5-methylcytosine, 5-hydrocymethylcytosine (HMC), glycosyl HMC andgentiobiosyl HMC, as well synthetic nucleobases, e.g., 2-aminoadenine,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N⁶(6-aminohexyl)adenine and2,6-diaminopurine (Kornberg, A., DNA Replication, W.H. Freeman & Co.,San Francisco, 1980, pages 75-77; Gebeyehu, G., et al., Nucleic AcidsRes., 1987, 15, 4513).

[0032] C. Sugar Modifications

[0033] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes an alkoxyalkoxy group, 2′-methoxyethoxy(2′-O—CH ₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE)(Martin et al., Helv. Chim. Acta, 1995, 78, 486-504). Further preferredmodifications include 2′-dimethylaminooxyethoxy, i.e., a2′-O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE and2′-dimethylaminoethoxyethoxy, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂.

[0034] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference.

[0035] D. Other Modifications

[0036] Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. The5′ and 3′ termini of an oligonucleotide may also be modified to serve aspoints of chemical conjugation of, e.g., lipophilic moieties (seeimmediately subsequent paragraph), intercalating agents (Kuyavin et al.,WO 96/32496, published Oct. 17, 1996; Nguyen et al., U.S. Pat. No.4,835,263, issued May 30, 1989) or hydroxyalkyl groups (Helene et al.,WO 96/34008, published Oct. 31, 1996).

[0037] Other positions within an oligonucleotide of the invention can beused to chemically link thereto one or more effector groups to form anoligonucleotide conjugate. An “effector group” is a chemical moiety thatis capable of carrying out a particular chemical or biological function.Examples of such effector groups include, but are not limited to, an RNAcleaving group, a reporter group, an intercalator, a group for improvingthe pharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide and othersubstituents having similar properties. A variety of chemical linkersmay be used to conjugate an effector group to an oligonucleotide of theinvention. As an example, U.S. Pat. No. 5,578,718 to Cook et al.discloses methods of attaching an alkylthio linker, which may be furtherderivatized to include additional groups, to ribofuranosyl positions,nucleosidic base positions, or on internucleoside linkages. Additionalmethods of conjugating oligonucleotides to various effector groups areknown in the art; see, e.g., Protocols for Oligonucleotide Conjugates(Methods in Molecular Biology, Volume 26) Agrawal, S., ed., HumanaPress, Totowa, N.J., 1994.

[0038] Another preferred additional or alternative modification of theoligonucleotides of the invention involves chemically linking to theoligonucleotide one or more lipophilic moieties which enhance thecellular uptake of the oligonucleotide. Such lipophilic moieties may belinked to an oligonucleotide at several different positions on theoligonucleotide. Some preferred positions include the 3′ position of thesugar of the 3′ terminal nucleotide, the 5′ position of the sugar of the5′ terminal nucleotide, and the 2′ position of the sugar of anynucleotide. The N⁶ position of a purine nucleobase may also be utilizedto link a lipophilic moiety to an oligonucleotide of the invention(Gebeyehu, G., et al., Nucleic Acids Res., 1987, 15, 4513). Suchlipophilic moieties include but are not limited to a cholesteryl moiety(Letsinger et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86, 6553),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053),a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y.Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov etal., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75,49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylaminocarbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923). Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides, are disclosed in U.S. Pat. Nos. 5,138,045, 5,218,105and 5,459,255.

[0039] The present invention also includes oligonucleotides that aresubstantially chirally pure with regard to particular positions withinthe oligonucleotides. Examples of substantially chirally pureoligonucleotides include, but are not limited to, those havingphosphorothioate linkages that are at least 75% Sp or Rp (Cook et al.,U.S. Pat. No. 5,587,361) and those having substantially chirally pure(Sp or Rp) alkylphosphonate, phosphoamidate or phosphotriester linkages(Cook, U.S. Pat. Nos. 5,212,295 and 5,521,302).

[0040] E. Chimeric Oligonucleotides

[0041] The present invention also includes oligonucleotides which arechimeric oligonucleotides. “Chimeric” oligonucleotides or “chimeras,” inthe context of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionwherein the oligonucleotide is modified so as to confer upon theoligonucleotide increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof antisense inhibition of gene expression. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art. Byway of example, such “chimeras” may be “gapmers,” i.e., oligonucleotidesin which a central portion (the “gap”) of the oligonucleotide serves asa substrate for, e.g., RNase H, and the 5′ and 3′ portions (the “wings”)are modified in such a fashion so as to have greater affinity for thetarget RNA molecule but are unable to support nuclease activity (e.g.,2′-fluoro- or 2′-methoxyethoxy-substituted). Other chimeras include“wingmers,” that is, oligonucleotides in which the 5′ portion of theoligonucleotide serves as a substrate for, e.g., RNase H, whereas the 3′portion is modified in such a fashion so as to have greater affinity forthe target RNA molecule but is unable to support nuclease activity(e.g., 2′-fluoro- or 2′-methoxyethoxy-substituted), or vice-versa.

[0042] F. Synthesis

[0043] The oligonucleotides used in accordance with this invention maybe conveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0044] 1. Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. patents or pendingpatent applications, each of which is commonly assigned with thisapplication: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamineconjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomersfor the preparation of oligonucleotides having chiral phosphoruslinkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn tooligonucleotides having modified backbones; U.S. Pat. No. 5,386,023,drawn to backbone modified oligonucleotides and the preparation thereofthrough reductive coupling; U.S. Pat. No. 5,457,191, drawn to modifiednucleobases based on the 3-deazapurine ring system and methods ofsynthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingE-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, issued Jun. 29, 1993, and 5,608,046, both drawn toconjugated 4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240,and 5,610,289, drawn to backbone modified oligonucleotide analogs; andU.S. patent application Ser. No. 08/383,666, filed Feb. 3, 1995, andU.S. Pat. No. 5,459,255, drawn to, inter alia, methods of synthesizing2′-fluoro-oligonucleotides.

[0045] 2. 5-methyl-cytosine: In 2′-methoxyethoxy-modifiedoligonucleotides, 5-methyl-2′-methoxyethoxy-cytosine residues are usedand are prepared as follows.

[0046] (a) 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]

[0047] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to ref lux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60? C. at 1mm Hg for 24 h) to give a solid which was crushed to a light tan powder(57 g, 85% crude yield). The material was used as is for furtherreactions.

[0048] (b) 2′-O-Methoxyethyl-5-methyluridine

[0049] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160? C. After heating for 48 hours at 155-160?C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product.

[0050] (c) 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0051] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/Hexane/Acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0052] (d)3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0053] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by tlc by first quenching the tlc sample with the additionof MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35? C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approximately90% product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane (4:1). Pure product fractions were evaporatedto yield 96 g (84%).

[0054] (e)3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0055] A first solution was prepared by dissolving3-O-acetyl-2′-O-methoxyethyl-5 ′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5? C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10? C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

[0056] (f) 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0057] A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) indioxane (500 mL) and NH₄OH (30 mL) was stirred at room temperature for 2hours. The dioxane solution was evaporated and the residue azeotropedwith MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) andtransferred to a 2 liter stainless steel pressure vessel. Methanol (400mL) saturated with NH₃ gas was added and the vessel heated to 100? C.for 2 hours (thin layer chromatography, tlc, showed completeconversion). The vessel contents were evaporated to dryness and theresidue was dissolved in EtOAc (500 mL) and washed once with saturatedNaCl (200 mL). The organics were dried over sodium sulfate and thesolvent was evaporated to give 85 g (95%) of the title compound.

[0058] (g)N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0059] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, tlc showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0060] (h)N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0061]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAcHexane (3:1) as theeluting solvent. The pure fractions were combined to give 90.6 g (87%)of the title compound.

3. 2′-O-(Aminooxyethyl)nucleoside Amidites and2′-O-(dimethylaminooxyethyl)nucleoside Amidites2′-(Dimethylaminooxyethoxy)nucleoside Amidites

[0062] 2′-(Dimethylaminooxyethoxy)nucleoside amidites [also known in theart as 2′-O-(dimethylaminooxyethyl)nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0063] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0064] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0065]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40? C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl -5-methyluridineas white foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0066]2-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10? C. to 0?C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl)thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%).

[0067]5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0068]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10? C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10? C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10? C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10? C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0069] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0070] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40? C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0071] 5-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40? C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy)nucleoside amidites

[0072] 2′-(Aminooxyethoxy)nucleoside amidites [also known in the art as2′-O-(aminooxyethyl)nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0073] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl)diaminopurine riboside alongwith a minor amount of the 3′-O-isomer.2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and convertedto 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may bedisplaced by N-hydroxyphthalimide via a Mitsunobu reaction, and theprotected nucleoside may phosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramiditel]

[0074] 2. Bioequivalents

[0075] The compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto “prodrugs” and “pharmaceutically acceptable salts” of theoligonucleotides of the invention, pharmaceutically acceptable salts ofsuch prodrugs, and other bioequivalents.

[0076] A. Oligonucleotide Prodrugs

[0077] The oligonucleotides of the invention may additionally oralternatively be prepared to be delivered in a “prodrug” form. The term“prodrug” indicates a therapeutic agent that is prepared in an inactiveform that is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993.

[0078] B. Pharmaceutically Acceptable Salts

[0079] The term Apharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of theoligonucleotides of the invention: i.e., salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0080] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1). The base addition salts of said acidic compounds areprepared by contacting the free acid form with a sufficient amount ofthe desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0081] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0082] 3. Exemplary Utilities of the Invention

[0083] The oligonucleotides of the present invention specificallyhybridize to nucleic acids (e.g., mRNAs) encoding a JNK protein. Theoligonucleotides of the present invention can be utilized as therapeuticcompounds, as diagnostic tools or research reagents that can beincorporated into kits, and in purifications and cellular productpreparations, as well as other methodologies, which are appreciated bypersons of ordinary skill in the art.

[0084] A. Assays and Diagnostic Applications

[0085] The oligonucleotides of the present invention can be used todetect the presence of JNK protein-specific nucleic acids in a cell ortissue sample. For example, radiolabeled oligonucleotides can beprepared by ³²P labeling at the 5′ end with polynucleotide kinase.(Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1989, Volume 2, pg. 10.59.) Radiolabeledoligonucleotides are then contacted with cell or tissue samplessuspected of containing JNK protein message RNAs (and thus JNKproteins), and the samples are washed to remove unbound oligonucleotide.Radioactivity remaining in the sample indicates the presence of boundoligonucleotide, which in turn indicates the presence of nucleic acidscomplementary to the oligonucleotide, and can be quantitated using ascintillation counter or other routine means. Expression of nucleicacids encoding these proteins is thus detected.

[0086] Radiolabeled oligonucleotides of the present invention can alsobe used to perform autoradiography of tissues to determine thelocalization, distribution and quantitation of JNK proteins forresearch, diagnostic or therapeutic purposes. In such studies, tissuesections are treated with radiolabeled oligonucleotide and washed asdescribed above, then exposed to photographic emulsion according toroutine autoradiography procedures. The emulsion, when developed, yieldsan image of silver grains over the regions expressing a JNK proteingene. Quantitation of the silver grains permits detection of theexpression of mRNA molecules encoding these proteins and permitstargeting of oligonucleotides to these areas.

[0087] Analogous assays for fluorescent detection of expression of JNKprotein nucleic acids can be developed using oligonucleotides of thepresent invention which are conjugated with fluorescein or otherfluorescent tags instead of radiolabeling. Such conjugations areroutinely accomplished during solid phase synthesis usingfluorescently-labeled amidites or controlled pore glass (CPG) columns.Fluorescein-labeled amidites and CPG are available from, e.g., GlenResearch, Sterling Va. Other means of labeling oligonucleotides areknown in the art (see, e.g., Ruth, Chapter 6 In: Methods in MolecularBiology, Vol. 26: Protocols for Oligonucleotide Conjugates, Agrawal,ed., Humana Press Inc., Totowa, N.J., 1994, pages 167-185).

[0088] Kits for detecting the presence or absence of expression of a JNKprotein may also be prepared. Such kits include an oligonucleotidetargeted to an appropriate gene, i.e., a gene encoding a JNK protein.Appropriate kit and assay formats, such as, e.g., “sandwich” assays, areknown in the art and can easily be adapted for use with theoligonucleotides of the invention. Hybridization of the oligonucleotidesof the invention with a nucleic acid encoding a JNK protein can bedetected by means known in the art. Such means may include conjugationof an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection systems.

[0089] B. Protein Purifications

[0090] The oligonucleotides of the invention are also useful for thepurification of specific Jun kinase proteins from cells that normallyexpress a set of JNK proteins which are similar to each other in termsof their polypeptide sequences and biochemical properties. As anexample, the purification of a JNK1 protein from cells that expressesJNK1, JNK2 and JNK3 proteins can be enhanced by first treating suchcells with oligonucleotides that inhibit the expression of JNK2 and JNK3and/or with oligonucleotides that increase the expression of JNK1,because such treatments will increase the relative ratio of JNK1relative to JNK2 and JNK3. As a result, the yield of JNK1 fromsubsequent purification steps will be improved as the amount of thebiochemically similar (and thus likely to contaminate) JNK2 and JNK3proteins in extracts prepared from cells so treated will be diminished.

[0091] C. Biologically Active Oligonucleotides

[0092] The invention is also drawn to the administration ofoligonucleotides having biological activity to cultured cells, isolatedtissues and organs and animals. By “having biological activity,” it ismeant that the oligonucleotide functions to modulate the expression ofone or more genes in cultured cells, isolated tissues or organs and/oranimals. Such modulation can be achieved by an antisense oligonucleotideby a variety of mechanisms known in the art, including but not limitedto transcriptional arrest; effects on RNA processing (capping,polyadenylation and splicing) and transportation; enhancement ofcellular degradation of the target nucleic acid; and translationalarrest (Crooke et al., Exp. Opin. Ther. Patents, 1996 6, 855).

[0093] In an animal other than a human, the compositions and methods ofthe invention can be used to study the function of one or more genes inthe animal. For example, antisense oligonucleotides have beensystemically administered to rats in order to study the role of theN-methyl-D-aspartate receptor in neuronal death, to mice in order toinvestigate the biological role of protein kinase C-a, and to rats inorder to examine the role of the neuropeptide Y1 receptor in anxiety(Wahlestedt et al., Nature, 1993, 363, 260; Dean et al., Proc. Natl.Acad. Sci. U.S.A., 1994, 91, 11762; and Wahlestedt et al., Science,1993, 259, 528, respectively). In instances where complex families ofrelated proteins are being investigated, “antisense knockouts” (i.e.,inhibition of a gene by systemic administration of antisenseoligonucleotides) may represent the most accurate means for examining aspecific member of the family (see, generally, Albert et al., TrendsPharmacol. Sci., 1994, 15, 250).

[0094] The compositions and methods of the invention also havetherapeutic uses in an animal, including a human, having (i.e.,suffering from), or known to be or suspected of being prone to having, adisease or disorder that is treatable in whole or in part with one ormore nucleic acids. The term “therapeutic uses” is intended to encompassprophylactic, palliative and curative uses wherein the oligonucleotidesof the invention are contacted with animal cells either in vivo or exvivo. When contacted with animal cells ex vivo, a therapeutic useincludes incorporating such cells into an animal after treatment withone or more oligonucleotides of the invention.

[0095] For therapeutic uses, an animal suspected of having a disease ordisorder which can be treated or prevented by modulating the expressionor activity of a JNK protein is, for example, treated by administeringoligonucleotides in accordance with this invention. The oligonucleotidesof the invention can be utilized in pharmaceutical compositions byadding an effective amount of an oligonucleotide to a suitablepharmaceutically acceptable carrier such as, e.g., a diluent. Workers inthe field have identified antisense, triplex and other oligonucleotidecompositions which are capable of modulating expression of genesimplicated in viral, fungal and metabolic diseases. Antisenseoligonucleotides have been safely administered to humans and severalclinical trials are presently underway. It is thus established thatoligonucleotides can be useful therapeutic instrumentalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans. The following U.S. patentsdemonstrate palliative, therapeutic and other methods utilizingantisense oligonucleotides. U.S. Pat. No. 5,135,917 provides antisenseoligonucleotides that inhibit human interleukin-1 receptor expression.U.S. Pat. No. 5,098,890 is directed to antisense oligonucleotidescomplementary to the c-myb oncogene and antisense oligonucleotidetherapies for certain cancerous conditions. U.S. Pat. No. 5,087,617provides methods for treating cancer patients with antisenseoligonucleotides. U.S. Pat. No. 5,166,195 provides oligonucleotideinhibitors of Human Immunodeficiency Virus (HIV). U.S. Pat. No.5,004,810 provides oligomers capable of hybridizing to herpes simplexvirus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428provides antisense oligonucleotides having antiviral activity againstinfluenza virus. U.S. Pat. No. 5,004,810 provides antisenseoligonucleotides and methods using them to inhibit HTLV-III replication.U.S. Pat. No. 5,286,717 provides oligonucleotides having a complementarybase sequence to a portion of an oncogene. U.S. Pat. Nos. 5,276,019 and5,264,423 are directed to phosphorothioate oligonucleotide analogs usedto prevent replication of foreign nucleic acids in cells. U.S. Pat. No.4,689,320 is directed to antisense oligonucleotides as antiviral agentsspecific to cytomegalovirus (CMV). U.S. Pat. No. 5,098,890 providesoligonucleotides complementary to at least a portion of the mRNAtranscript of the human c-myb gene. U.S. Pat. No. 5,242,906 providesantisense oligonucleotides useful in the treatment of latentEpstein-Barr virus (EBV) infections.

[0096] As used herein, the term “disease or disorder” (1) includes anyabnormal condition of an organism or part, especially as a consequenceof infection, inherent weakness, environmental stress, that impairsnormal physiological functioning; (2) excludes pregnancy per se but notautoimmune and other diseases associated with pregnancy; and (3)includes cancers and tumors. The term “known to be or suspected of beingprone to having a disease or disorder” indicates that the subject animalhas been determined to be, or is suspected of being, at increased risk,relative to the general population of such animals, of developing aparticular disease or disorder as herein defined. For example, a subjectanimal “known to be or suspected of being prone to having a disease ordisorder” could have a personal and/or family medical history thatincludes frequent occurrences of a particular disease or disorder. Asanother example, a subject animal “known to be or suspected of beingprone to having a disease or disorder” could have had such asusceptibility determined by genetic screening according to techniquesknown in the art (see, e.g., U.S. Congress, Office of TechnologyAssessment, Chapter 5 In: Genetic Monitoring and Screening in theWorkplace, OTA-BA-455, U.S. Government Printing Office, Washington,D.C., 1990, pages 75-99). The term “a disease or disorder that istreatable in whole or in part with one or more nucleic acids” refers toa disease or disorder, as herein defined, (1) the management, modulationor treatment thereof, and/or (2) therapeutic, curative, palliativeand/or prophylactic relief therefrom, can be provided via theadministration of an antisense oligonucleotide.

[0097] 4. Pharmaceutical Compositions

[0098] The formulation of pharmaceutical compositions comprising theoligonucleotides of the invention, and their subsequent administration,are believed to be within the skill of those in the art.

[0099] A. Therapeutic Considerations

[0100] In general, for therapeutic applications, a patient (i.e., ananimal, including a human, having or predisposed to a disease ordisorder) is administered one or more oligonucleotides, in accordancewith the invention in a pharmaceutically acceptable carrier in dosesranging from 0.01 μg to 100 g per kg of body weight depending on the ageof the patient and the severity of the disorder or disease state beingtreated. Further, the treatment regimen may last for a period of timewhich will vary depending upon the nature of the particular disease ordisorder, its severity and the overall condition of the patient, and mayextend from once daily to once every 20 years. In the context of theinvention, the term “treatment regimen” is meant to encompasstherapeutic, palliative and prophylactic modalities. Followingtreatment, the patient is monitored for changes in his/her condition andfor alleviation of the symptoms of the disorder or disease state. Thedosage of the nucleic acid may either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the disorderor disease state is observed, or if the disorder or disease state hasbeen ablated.

[0101] Dosing is dependent on severity and responsiveness of the diseasestate to be treated, with the course of treatment lasting from severaldays to several months, or until a cure is effected or a diminution ofthe disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual oligonucleotides, andcan generally be estimated based on EC₅₀s found to be effective in invitro and in vivo animal models. In general, dosage is from 0.01 μg to100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. An optimaldosing schedule is used to deliver a therapeutically effective amount ofthe oligonucleotide being administered via a particular mode ofadministration.

[0102] The term “therapeutically effective amount,” for the purposes ofthe invention, refers to the amount of oligonucleotide-containingpharmaceutical composition which is effective to achieve an intendedpurpose without undesirable side effects (such as toxicity, irritationor allergic response). Although individual needs may vary, determinationof optimal ranges for effective amounts of pharmaceutical compositionsis within the skill of the art. Human doses can be extrapolated fromanimal studies (Katocs et al., Chapter 27 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990). Generally, the dosage required to provide an effective amount ofa pharmaceutical composition, which can be adjusted by one skilled inthe art, will vary depending on the age, health, physical condition,weight, type and extent of the disease or disorder of the recipient,frequency of treatment, the nature of concurrent therapy (if any) andthe nature and scope of the desired effect(s) (Nies et al., Chapter 3In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al., Eds., McGraw-Hill, New York, N.Y., 1996).

[0103] As used herein, the term “high risk individual” is meant to referto an individual for whom it has been determined, via, e.g., individualor family history or genetic testing, has a significantly higher thannormal probability of being susceptible to the onset or recurrence of adisease or disorder. As art of treatment regimen for a high riskindividual, the individual can be prophylactically treated to preventthe onset or recurrence of the disease or disorder. The term“prophylactically effective amount” is meant to refer to an amount of apharmaceutical composition which produces an effect observed as theprevention of the onset or recurrence of a disease or disorder.Prophylactically effective amounts of a pharmaceutical composition aretypically determined by the effect they have compared to the effectobserved when a second pharmaceutical composition lacking the activeagent is administered to a similarly situated individual.

[0104] Following successful treatment, it may be desirable to have thepatient undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the nucleic acid is administered in maintenancedoses, ranging from 0.01 μg to 100 g per kg of body weight, once or moredaily, to once every 20 years. For example, in the case of in individualknown or suspected of being prone to an autoimmune or inflammatorycondition, prophylactic effects may be achieved by administration ofpreventative doses, ranging from 0.01 μg to 100 g per kg of body weight,once or more daily, to once every 20 years. In like fashion, anindividual may be made less susceptible to an inflammatory conditionthat is expected to occur as a result of some medical treatment, e.g.,graft versus host disease resulting from the transplantation of cells,tissue or an organ into the individual.

[0105] In some cases it may be more effective to treat a patient with anoligonucleotide of the invention in conjunction with other traditionaltherapeutic modalities in order to increase the efficacy of a treatmentregimen. In the context of the invention, the term “A treatment regimen”is meant to encompass therapeutic, palliative and prophylacticmodalities. For example, a patient may be treated with conventionalchemotherapeutic agents, particularly those used for tumor and cancertreatment. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, daunomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 1206-1228, Berkow et al., Eds.,Rahay, N.J., 1987). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

[0106] In another preferred embodiment of the invention, a firstantisense oligonucleotide targeted to a first JNK protein is used incombination with a second antisense oligonucleotide targeted to a secondJNK protein in order to such JNK proteins to a more extensive degreethan can be achieved when either oligonucleotide is used individually.In various embodiments of the invention, the first and second JNKproteins which are targeted by such oligonucleotides are identical, aredifferent JNK proteins or are different isoforms of the same JNKprotein.

[0107] B. Pharmaceutical Compositions

[0108] Pharmaceutical compositions for the non-parenteral administrationof oligonucleotides may include sterile aqueous solutions which may alsocontain buffers, diluents and other suitable additives. Pharmaceuticallyacceptable organic or inorganic carrier substances suitable fornon-parenteral administration which do not deleteriously react witholigonucleotides can be used. Suitable pharmaceutically acceptablecarriers include, but are not limited to, water, salt solutions,alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesiumstearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like. The pharmaceutical compositions canbe sterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings flavoringsand/or aromatic substances and the like which do not deleteriously reactwith the oligonucleotide(s) of the pharmaceutical composition.Pharmaceutical compositions in the form of aqueous suspensions maycontain substances which increase the viscosity of the suspensionincluding, for example, sodium carboxymethylcellulose, sorbitol and/ordextran. Optionally, such suspensions may also contain stabilizers.

[0109] In one embodiment of the invention, an oligonucleotide isadministered via the rectal mode. In particular, pharmaceuticalcompositions for rectal administration include foams, solutions (enemas)and suppositories. Rectal suppositories for adults are usually taperedat one or both ends and typically weigh about 2 g each, with infantrectal suppositories typically weighing about one-half as much, when theusual base, cocoa butter, is used (Block, Chapter 87 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990).

[0110] In a preferred embodiment of the invention, one or moreoligonucleotides are administered via oral delivery. Pharmaceuticalcompositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, troches, tablets or SECs (soft elastic capsules or “caplets”).Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids,carrier substances or binders may be desirably added to suchpharmaceutical compositions. The use of such pharmaceutical compositionshas the effect of delivering the oligonucleotide to the alimentary canalfor exposure to the mucosa thereof. Accordingly, the pharmaceuticalcomposition can comprise material effective in protecting theoligonucleotide from pH extremes of the stomach, or in releasing theoligonucleotide over time, to optimize the delivery thereof to aparticular mucosal site. Enteric coatings for acid-resistant tablets,capsules and caplets are known in the art and typically include acetatephthalate, propylene glycol and sorbitan monoleate.

[0111] Various methods for producing pharmaceutical compositions foralimentary delivery are well known in the art. See, generally, Nairn,Chapter 83; Block, Chapter 87; Rudnic et al., Chapter 89; Porter,Chapter 90; and Longer et al., Chapter 91 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990. The oligonucleotides of the invention can be incorporated in aknown manner into customary pharmaceutical compositions, such astablets, coated tablets, pills, granules, aerosols, syrups, emulsions,suspensions and solutions, using inert, non-toxic, pharmaceuticallyacceptable carriers (excipients). The therapeutically active compoundshould in each case be present here in a concentration of about 0.5% toabout 95% by weight of the total mixture, i.e., in amounts which aresufficient to achieve the stated dosage range. The pharmaceuticalcompositions are prepared, for example, by diluting the active compoundswith pharmaceutically acceptable carriers, if appropriate usingemulsifying agents and/or dispersing agents, and, for example, in thecase where water is used as the diluent, organic solvents can be used asauxiliary solvents if appropriate. Pharmaceutical compositions may beformulated in a conventional manner using additional pharmaceuticallyacceptable carriers as appropriate. Thus, the compositions may beprepared by conventional means with additional excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrates (e.g., starchor sodium starch glycolate); or wetting agents (e.g., sodium laurylsulfate). Tablets may be coated by methods well known in the art. Thepreparations may also contain flavoring, coloring and/or sweeteningagents as appropriate.

[0112] The pharmaceutical compositions, which may conveniently bepresented in unit dosage form, may be prepared according to conventionaltechniques well known in the pharmaceutical industry. Such techniquesinclude the step of bringing into association the active ingredient(s)with the pharmaceutically acceptable carrier(s). In general thepharmaceutical compositions are prepared by uniformly and intimatelybringing into association the active ingredient(s) with liquidexcipients or finely divided solid excipients or both, and then, ifnecessary, shaping the product.

[0113] Pharmaceutical compositions of the present invention suitable fororal administration may be presented as discrete units such as capsules,cachets or tablets each containing predetermined amounts of the activeingredients; as powders or granules; as solutions or suspensions in anaqueous liquid or a non-aqueous liquid; or as oil-in-water emulsions orwater-in-oil liquid emulsions. A tablet may be made by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine, the activeingredients in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, preservative,surface active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredients therein. Pharmaceuticalcompositions for parenteral, intrathecal or intraventricularadministration, or colloidal dispersion systems, may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives.

[0114] C. Penetration Enhancers

[0115] Pharmaceutical compositions comprising the oligonucleotides ofthe present invention may also include penetration enhancers in order toenhance the alimentary delivery of the oligonucleotides. Penetrationenhancers may be classified as belonging to one of five broadcategories, i.e., fatty acids, bile salts, chelating agents, surfactantsand non-surfactants (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, 8, 91-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1).

[0116] 1. Fatty Acids

[0117] Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid,linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651).

[0118] 2. Bile Salts

[0119] The physiological roles of bile include the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., Eds., McGraw-Hill, New York,N.Y., 1996, pages 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus, the term“bile salt” includes any of the naturally occurring components of bileas well as any of their synthetic derivatives.

[0120] 3. Chelating Agents

[0121] Chelating agents have the added advantage of also serving asDNase inhibitors and include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. ControlRel., 1990, 14, 43).

[0122] 4. Surfactants

[0123] Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al.,J. Pharm. Phamacol., 1988, 40, 252).

[0124] 5. Non-Surfactants

[0125] Non-surfactants include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621).

[0126] D. Carrier Compounds

[0127] As used herein, “carrier compound” refers to a nucleic acid, oranalog thereof, which is inert (i.e., does not possess biologicalactivity per se) but is recognized as a nucleic acid by in vivoprocesses that reduce the bioavailability of a nucleic acid havingbiological activity by, for example, degrading the biologically activenucleic acid or promoting its removal from circulation. Thecoadministration of a nucleic acid and a carrier compound, typicallywith an excess of the latter substance, can result in a substantialreduction of the amount of nucleic acid recovered in the liver, kidneyor other extracirculatory reservoirs, presumably due to competitionbetween the carrier compound and the nucleic acid for a common receptor.For example, the recovery of a partially phosphorothioatedoligonucleotide in hepatic tissue is reduced when it is coadministeredwith polyinosinic acid, dextran sulfate, polycytidic acid or4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense & Nucl.Acid Drug Dev., 1996, 6, 177).

[0128] E. Pharmaceutically Acceptable Carriers

[0129] In contrast to a carrier compound, a “pharmaceutically acceptablecarrier” (excipient) is a pharmaceutically acceptable solvent,suspending agent or any other pharmacologically inert vehicle fordelivering one or more nucleic acids to an animal. The pharmaceuticallyacceptable carrier may be liquid or solid and is selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, etc., when combined with a nucleic acid andthe other components of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers include, but are not limited to,binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate,etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidalsilicon dioxide, stearic acid, metallic stearates, hydrogenatedvegetable oils, corn starch, polyethylene glycols, sodium benzoate,sodium acetate, etc.); disintegrates (e.g., starch, sodium starchglycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate,etc.). Sustained release oral delivery systems and/or enteric coatingsfor orally administered dosage forms are described in U.S. Pat. Nos.4,704,295; 4,556,552; 4,309,406; and 4,309,404.

[0130] F. Miscellaneous Additional Components

[0131] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional compatiblepharmaceutically-active materials such as, e.g., antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the composition of present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the invention.

[0132] G. Colloidal Dispersion Systems

[0133] Regardless of the method by which the oligonucleotides of theinvention are introduced into a patient, colloidal dispersion systemsmay be used as delivery vehicles to enhance the in vivo stability of theoligonucleotides and/or to target the oligonucleotides to a particularorgan, tissue or cell type. Colloidal dispersion systems include, butare not limited to, macromolecule complexes, nanocapsules, microspheres,beads and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles and liposomes. A preferred colloidal dispersionsystem is a plurality of liposomes, artificial membrane vesicles whichmay be used as cellular delivery vehicles for bioactive agents in vitroand in vivo (Mannino et al., Biotechniques, 1988, 6, 682; Blume andCevc, Biochem. et Biophys. Acta, 1990, 1029, 91; Lappalainen et al.,Antiviral Res., 1994, 23, 119; Chonn and Cullis, Current Op. Biotech.,1995, 6, 698). It has been shown that large unilamellar vesicles (LUV),which range in size from 0.2-0.4 μm, can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand delivered to brain cells in a biologically active form (Fraley etal., Trends Biochem. Sci., 1981, 6, 77). The composition of the liposomeis usually a combination of lipids, particularly phospholipids, inparticular, high phase transition temperature phospholipids, usually incombination with one or more steroids, particularly cholesterol.Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, sphingolipids, phosphatidylethanolamine,cerebrosides and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbonatoms, particularly from 16-18 carbon atoms, and is saturated (lackingdouble bonds within the 14-18 carbon atom chain). Illustrativephospholipids include phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0134] The targeting of colloidal dispersion systems, includingliposomes, can be either passive or active. Passive targeting utilizesthe natural tendency of liposomes to distribute to cells of thereticuloendothelial system in organs that contain sinusoidalcapillaries. Active targeting, by contrast, involves modification of theliposome by coupling thereto a specific ligand such as a viral proteincoat (Morishita et al., Proc. Natl. Acad. Sci. (U.S.A.), 1993, 90,8474), monoclonal antibody (or a suitable binding portion thereof),sugar, glycolipid or protein (or a suitable oligopeptide fragmentthereof), or by changing the composition and/or size of the liposome inorder to achieve distribution to organs and cell types other than thenaturally occurring sites of localization. The surface of the targetedcolloidal dispersion system can be modified in a variety of ways. In thecase of a liposomal targeted delivery system, lipid groups can beincorporated into the lipid bilayer of the liposome in order to maintainthe targeting ligand in close association with the lipid bilayer.Various linking groups can be used for joining the lipid chains to thetargeting ligand. The targeting ligand, which binds a specific cellsurface molecule found predominantly on cells to which delivery of theoligonucleotides of the invention is desired, may be, for example, (1) ahormone, growth factor or a suitable oligopeptide fragment thereof whichis bound by a specific cellular receptor predominantly expressed bycells to which delivery is desired or (2) a polyclonal or monoclonalantibody, or a suitable fragment thereof (e.g., Fab; F(ab′)₂) whichspecifically binds an antigenic epitope found predominantly on targetedcells. Two or more bioactive agents (e.g., an oligonucleotide and aconventional drug; two oligonucleotides) can be combined within, anddelivered by, a single liposome. It is also possible to add agents tocolloidal dispersion systems which enhance the intercellular stabilityand/or targeting of the contents thereof.

[0135] 5. Means of Administration

[0136] The present invention provides compositions comprisingoligonucleotides intended for administration to an animal. For purposesof the invention, unless otherwise specified, the term “animal” is meantto encompass humans as well as other mammals, as well as reptiles,amphibians, and birds.

[0137] A. Parenteral Delivery

[0138] The term “parenteral delivery” refers to the administration of anoligonucleotide of the invention to an animal in a manner other thanthrough the digestive canal. Means of preparing and administeringparenteral pharmaceutical compositions are known in the art (see, e.g.,Avis, Chapter 84 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 1545-1569).Parenteral means of delivery include, but are not limited to, thefollowing illustrative examples.

[0139] 1. Intravitreal injection, for the direct delivery of drug to thevitreous humor of a mammalian eye, is described in U.S. Pat. No.5,591,720, the contents of which are hereby incorporated by reference.Means of preparing and administering ophthalmic preparations are knownin the art (see, e.g., Mullins et al., Chapter 86 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, pages 1581-1595).

[0140] 2. Intravenous administration of antisense oligonucleotides tovarious non-human mammals has been described by Iversen (Chapter 26 In:Antisense Research and Applications, Crooke et al., Eds., CBC Press,Boca Raton, Fla., 1993, pages 461-469). Systemic delivery ofoligonucleotides to non-human mammals via intraperitoneal means has alsobeen described (Dean et al., Proc. Natl. Acad. Sci. (U.S.A.), 1994, 91,11766).

[0141] 3. Intraluminal drug administration, for the direct delivery ofdrug to an isolated portion of a tubular organ or tissue (e.g., such asan artery, vein, ureter or urethra), may be desired for the treatment ofpatients with diseases or conditions afflicting the lumen of such organsor tissues. To effect this mode of oligonucleotide administration, acatheter or cannula is surgically introduced by appropriate means. Forexample, for treatment of the left common carotid artery, a cannula isinserted thereinto via the external carotid artery. After isolation of aportion of the tubular organ or tissue for which treatment is sought, acomposition comprising the oligonucleotides of the invention is infusedthrough the cannula or catheter into the isolated segment. Afterincubation for from about 1 to about 120 minutes, during which theoligonucleotide is taken up by cells of the interior lumen of thevessel, the infusion cannula or catheter is removed and flow within thetubular organ or tissue is restored by removal of the ligatures whicheffected the isolation of a segment thereof (Morishita et al., Proc.Natl. Acad. Sci. U.S.A., 1993, 90, 8474). Antisense oligonucleotides mayalso be combined with a biocompatible matrix, such as a hydrogelmaterial, and applied directly to vascular tissue in vivo (Rosenberg etal., U.S. Pat. No. 5,593,974, issued Jan. 14, 1997).

[0142] 4. Intraventricular drug administration, for the direct deliveryof drug to the brain of a patient, may be desired for the treatment ofpatients with diseases or conditions afflicting the brain. To effectthis mode of oligonucleotide administration, a silicon catheter issurgically introduced into a ventricle of the brain of a human patient,and is connected to a subcutaneous infusion pump (Medtronic Inc.,Minneapolis, Minn.) that has been surgically implanted in the abdominalregion (Zimm et al., Cancer Research, 1984, 44, 1698; Shaw, Cancer,1993, 72(11 Suppl.), 3416). The pump is used to inject theoligonucleotides and allows precise dosage adjustments and variation indosage schedules with the aid of an external programming device. Thereservoir capacity of the pump is 18-20 mL and infusion rates may rangefrom 0.1 mL/h to 1 mL/h. Depending on the frequency of administration,ranging from daily to monthly, and the dose of drug to be administered,ranging from 0.01 μg to 100 g per kg of body weight, the pump reservoirmay be refilled at 3-10 week intervals. Refilling of the pump isaccomplished by percutaneous puncture of the self-sealing septum of thepump.

[0143] 5. Intrathecal drug administration, for the introduction of adrug into the spinal column of a patient may be desired for thetreatment of patients with diseases of the central nervous system. Toeffect this route of oligonucleotide administration, a silicon catheteris surgically implanted into the L3-4 lumbar spinal interspace of ahuman patient, and is connected to a subcutaneous infusion pump whichhas been surgically implanted in the upper abdominal region (Luer andHatton, The Annals of Pharracotherapy, 1993, 27, 912; Ettinger et al.,Cancer, 1978, 41, 1270; Yaida et al., Regul. Pept., 1995, 59, 193). Thepump is used to inject the oligonucleotides and allows precise dosageadjustments and variations in dose schedules with the aid of an externalprogramming device. The reservoir capacity of the pump is 18-20 mL, andinfusion rates may vary from 0.1 mL/h to 1 mL/h. Depending on thefrequency of drug administration, ranging from daily to monthly, anddosage of drug to be administered, ranging from 0.01 μg to 100 g per kgof body weight, the pump reservoir may be refilled at 3-10 weekintervals. Refilling of the pump is accomplished by a singlepercutaneous puncture to the self-sealing septum of the pump. Thedistribution, stability and pharmacokinetics of oligonucleotides withinthe central nervous system may be followed according to known methods(Whitesell et al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 4665).

[0144] To effect delivery of oligonucleotides to areas other than thebrain or spinal column via this method, the silicon catheter isconfigured to connect the subcutaneous infusion pump to, e.g., thehepatic artery, for delivery to the liver (Kemeny et al., Cancer, 1993,71, 1964). Infusion pumps may also be used to effect systemic deliveryof oligonucleotides (Ewel et al., Cancer Research, 1992, 52, 3005;Rubenstein et al., J. Surg. Oncol., 1996, 62, 194).

[0145] 6. Epidermal and Transdermal Delivery, in which pharmaceuticalcompositions containing drugs are applied topically, can be used toadminister drugs to be absorbed by the local dermis or for furtherpenetration and absorption by underlying tissues, respectively. Means ofpreparing and administering medications topically are known in the art(see, e.g., Block, Chapter 87 In: Remington's Pharmaceutical Sciences,18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages1596-1609).

[0146] 7. Vaginal Delivery provides local treatment and avoids firstpass metabolism, degradation by digestive enzymes, and potentialsystemic side-effects. This mode of administration may be preferred forantisense oligonucleotides targeted to pathogenic organisms for whichthe vagina is the usual habitat, e.g., Trichomonas vaginalis. In anotherembodiment, antisense oligonucleotides to genes encoding sperm-specificantibodies can be delivered by this mode of administration in order toincrease the probability of conception and subsequent pregnancy. Vaginalsuppositories (Block, Chapter 87 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990, pages 1609-1614) or topical ointments can be used to effect thismode of delivery.

[0147] 8. Intravesical Delivery provides local treatment and avoidsfirst pass metabolism, degradation by digestive enzymes, and potentialsystemic side-effects. However, the method requires urethralcatheterization of the patient and a skilled staff. Nevertheless, thismode of administration may be preferred for antisense oligonucleotidestargeted to pathogenic organisms, such as T. vaginalis, which may invadethe urogenital tract.

[0148] B. Alimentary Delivery

[0149] The term “alimentary delivery” refers to the administration,directly or otherwise, to a portion of the alimentary canal of ananimal. The term “alimentary canal” refers to the tubular passage in ananimal that functions in the digestion and absorption of food and theelimination of food residue, which runs from the mouth to the anus, andany and all of its portions or segments, e.g., the oral cavity, theesophagus, the stomach, the small and large intestines and the colon, aswell as compound portions thereof such as, e.g., the gastro-intestinaltract. Thus, the term “alimentary delivery” encompasses several routesof administration including, but not limited to, oral, rectal,endoscopic and sublingual/buccal administration. A common requirementfor these modes of administration is absorption over some portion or allof the alimentary tract and a need for efficient mucosal penetration ofthe nucleic acid(s) so administered.

[0150] 1. Buccal/Sublingual Administration: Delivery of a drug via theoral mucosa has several desirable features, including, in manyinstances, a more rapid rise in plasma concentration of the drug thanvia oral delivery (Harvey, Chapter 35 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990, page 711). Furthermore, because venous drainage from the mouth isto the superior vena cava, this route also bypasses rapid first-passmetabolism by the liver. Both of these features contribute to thesublingual route being the mode of choice for nitroglycerin (Benet etal., Chapter 1 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., Eds., McGraw-Hill, New York,N.Y., 1996, page 7).

[0151] 2. Endoscopic Administration: Endoscopy can be used for drugdelivery directly to an interior portion of the alimentary tract. Forexample, endoscopic retrograde cystopancreatography (ERCP) takesadvantage of extended gastroscopy and permits selective access to thebiliary tract and the pancreatic duct (Hirahata et al., Gan To KagakuRyoho, 1992, 19(10 Suppl.), 1591). However, the procedure is unpleasantfor the patient, and requires a highly skilled staff.

[0152] 3. Rectal Administration: Drugs administered by the oral routecan often be alternatively administered by the lower enteral route,i.e., through the anal portal into the rectum or lower intestine. Rectalsuppositories, retention enemas or rectal catheters can be used for thispurpose and may be preferred when patient compliance might a otherwisebe difficult to achieve (e.g., in pediatric and geriatric applications,or when the patient is vomiting or unconscious). Rectal administrationmay result in more prompt and higher blood levels than the oral route,but the converse may be true as well (Harvey, Chapter 35 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, page 711). Because about 50% of the drug that isabsorbed from the rectum will bypass the liver, administration by thisroute significantly reduces the potential for first-pass metabolism(Benet et al., Chapter 1 In: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al., Eds., McGraw-Hill, NewYork, N.Y., 1996). 4. Oral Administration: The preferred method ofadministration is oral delivery, which is typically the most convenientroute for access to the systemic circulation. Absorption from thealimentary canal is governed by factors that are generally applicable,e.g., surface area for absorption, blood flow to the site of absorption,the physical state of the drug and its concentration at the site ofabsorption (Benet et al., Chapter 1 In: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al., Eds.,McGraw-Hill, New York, N.Y., 1996, pages 5-7). A significant factorwhich may limit the oral bioavailability of a drug is the degree of“first pass effects.” For example, some substances have such a rapidhepatic uptake that only a fraction of the material absorbed enters theperipheral blood (Van Berge-Henegouwen et al., Gastroenterology, 1977,73, 300). The compositions and methods of the invention circumvent, atleast partially, such first pass effects by providing improved uptake ofnucleic acids and thereby, e.g., causing the hepatic uptake system tobecome saturated and allowing a significant portion of the nucleic acidso administered to reach the peripheral circulation. Additionally oralternatively, the hepatic uptake system is saturated with one or moreinactive carrier compounds prior to administration of the active nucleicacid.

[0153] The following examples illustrate the invention and are notintended to limit the same. Those skilled in the art will recognize, orbe able to ascertain through routine experimentation, numerousequivalents to the specific substances and procedures described herein.Such equivalents are considered to be within the scope of the presentinvention.

EXAMPLES Example 1 Synthesis of Oligonucleotides

[0154] A. General Synthetic Techniques

[0155] Oligonucleotides were synthesized on an automated DNA synthesizerusing standard phosphoramidite chemistry with oxidation using iodine.β-Cyanoethyldiisopropyl phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of3H-1,2-benzodithiole-3-one-1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages.

[0156] The synthesis of 2′-O-methyl-(a.k.a. 2′-methoxy-)phosphorothioateoligonucleotides is according to the procedures set forth abovesubstituting 2′-O-methyl β-cyanoethyldiisopropyl phosphoramidites(Chemgenes, Needham, Mass.) for standard phosphoramidites and increasingthe wait cycle after the pulse delivery of tetrazole and base to 360seconds.

[0157] Similarly, 2′-O-propyl-(a.k.a 2′-propoxy-)phosphorothioateoligonucleotides are prepared by slight modifications of this procedureand essentially according to procedures disclosed in U.S. patentapplication Ser. No. 08/383,666, filed Feb. 3, 1995, which is assignedto the same assignee as the instant application.

[0158] The 2′-fluoro-phosphorothioate oligonucleotides of the inventionare synthesized using 5′-dimethoxytrityl-3′-phosphoramidites andprepared as disclosed in U.S. patent application Ser. No. 08/383,666,filed Feb. 3, 1995, and U.S. Pat. No. 5,459,255, which issued Oct. 8,1996, both of which are assigned to the same assignee as the instantapplication. The 2′-fluoro-oligonucleotides were prepared usingphosphoramidite chemistry and a slight modification of the standard DNAsynthesis protocol (i.e., deprotection was effected using methanolicammonia at room temperature).

[0159] The 2′-methoxyethoxy oligonucleotides were synthesizedessentially according to the methods of Martin et al. (Helv. Chim. Acta,1995, 78, 486). For ease of synthesis, the 3′ nucleotide of the2′-methoxyethoxy oligonucleotides was a deoxynucleotide, and2′-O—CH₂CH₂OCH₃cytosines were 5-methyl cytosines, which were synthesizedaccording to the procedures described below.

[0160] PNA antisense analogs are prepared essentially as described inU.S. Pat. Nos. 5,539,082 and 5,539,083, both of which (1) issued Jul.23, 1996, and (2) are assigned to the same assignee as the instantapplication.

[0161] B. Purification

[0162] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55? C.for 18 hours, the oligonucleotides were purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gelelectrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mMTris-borate buffer, pH 7.0. Oligodeoxynucleotides and theirphosphorothioate analogs were judged from electrophoresis to be greaterthan 80% full length material.

Example 2 Assays for Oligonucleotide-Mediated Inhibition of JNK mRNAExpression in Human Tumor Cells

[0163] In order to evaluate the activity of potential JNK-modulatingoligonucleotides, human lung carcinoma cell line A549 (American TypeCulture Collection, Rockville, Md. No. ATCC CCL-185) cells or other celllines as indicated in the Examples, were grown and treated witholigonucleotides or control solutions as detailed below. Afterharvesting, cellular extracts were prepared and examined for specificJNK mRNA levels or JNK protein levels (i.e., Northern or Western assays,respectively). In all cases, “% expression” refers to the amount ofJNK-specific signal in an oligonucleotide-treated cell relative to anuntreated cell (or a cell treated with a control solution that lacksoligonucleotide), and “% inhibition” is calculated as

100%−% Expression=% Inhibition.

[0164] Northern Assays

[0165] The mRNA expression of each JNK protein was determined by using anucleic acid probe specifically hybridizable thereto. Nucleic acidprobes specific for JNK1, JNK2 and JNK3 are described in Examples 3, 4and 5, respectively. The probes were radiolabelled by means well knownin the art (see, e.g., Short Protocols in Molecular Biology, 2nd Ed.,Ausubel et al., Eds., John Wiley & Sons, New York, 1992, pages 3-11 to2-3-44 and 4-17 to 4-18; Ruth, Chapter 6 In: Methods in MolecularBiology, Vol. 26: Protocols for Oligonucleotide Conjugates, Agrawal,ed., Humana Press Inc., Totowa, N.J., 1994, pages 167-185; and Chapter10 In: Molecular Cloning: A Laboratory Manual, 2nd Ed., Sambrook et al.,Eds., pages 10.1-10.70). The blots were stripped and reprobed with a³²P-labeled glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe(Clontech Laboratories, Inc., Palo Alto, Calif.) in order to confirmequal loading of RNA and to allow the levels of JNK transcripts to benormalized with regard to the G3PDH signals.

[0166] A549 cells were grown in T-75 flasks until 80-90% confluent. Atthis time, the cells were washed twice with 10 mL of media (DMEM),followed by the addition of 5 mL of DMEM containing 20 μg/mL ofLIPOFECTIN™ (i.e., 1:1 (w/w) DOTMA/DOPE, Life Technologies,Gaithersburg, Md.;DOTMA=N-[1-(2,3-dioleyoxy)propyl]-N,N,N-trimethylammonium chloride;DOPE=dioleoyl phosphatidylethanolamine). The oligonucleotides were addedfrom a 10 μM stock solution to a final concentration of 400 nM, and thetwo solutions were mixed by swirling the flasks. As a control, cellswere treated with LIPOFECTIN™ without oligonucleotide under the sameconditions and for the same times as the oligonucleotide-treatedsamples. After 4 hours at 37° C., the medium was replaced with freshDMEM containing 10% serum. The cells were allowed to recover for 18hours. Total cellular RNA was then extracted in guanidinium, subject togel electrophoresis and transferred to a filter according to techniquesknown in the art (see, e.g., Chapter 7 In: Molecular Cloning: ALaboratory Manual, 2nd Ed., Sambrook et al., Eds., pages 7.1-7.87, andShort Protocols in Molecular Biology, 2nd Ed., Ausubel et al., Eds.,John Wiley & Sons, New York, 1992, pages 2-24 to 2-30 and 4-14 to 4-29).Filters were typically hybridized overnight to a probe specific for theparticular JNK gene of interest in hybridization buffer (25 mM KPO₄, pH7.4; 5×SSC; 5×Denhardt's solution, 100 μg/ml Salmon sperm DNA and 50%formamide) (Alahari et al., Nucl. Acids Res., 1993, 21, 4079). This wasfollowed by two washes with 1×SSC, 0.1% SDS and two washes with0.25×SSC, 0.1% SDS. Hybridizing bands were visualized by exposure toX-OMAT AR film and quantitated using a PHOSPHORIMAGER™ essentiallyaccording to the manufacturer's instructions (Molecular Dynamics,Sunnyvale, Calif.).

[0167] Western Assays

[0168] A549 cells were grown and treated with oligonucleotides asdescribed above. Cells were lysed, and protein extracts wereelectrophoresed (SDS-PAGE) and transferred to nitrocellulose filters bymeans known in the art (see, e.g., Chapter 18 In: Molecular Cloning: ALaboratory Manual, 2nd Ed., Sambrook et al., Eds., pages 18.34,18.47-18.54 and 18.60-18.75)). The amount of each JNK protein wasdetermined by using a primary antibody that specifically recognizes theappropriate JNK protein. The primary antibodies specific for each JNKprotein are described in the appropriate Examples. The primaryantibodies were detected by means well known in the art (see, e.g.,Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., Eds.,John Wiley & Sons, New York, 1992, pages 10-33 to 10-35; and Chapter 18In: Molecular Cloning: A Laboratory Manual, 2nd Ed., Sambrook et al.,Eds., pages 18.1-18.75 and 18.86-18.88) and quantitated using aPHOSPHORIMAGER™ essentially according to the manufacturer's instructions(Molecular Dynamics, Sunnyvale, Calif.).

[0169] Levels of JNK proteins can also be quantitated by measuring thelevel of their corresponding kinase activity. Such kinase assays can bedone in gels in situ (Hibi et al., Genes & Dev., 1993, 7, 2135) or afterimmunoprecipitation from cellular extracts (Derijard et al., Cell, 1994,76, 1025). Substrates and/or kits for such assays are commerciallyavailable from, for example, Upstate Biotechnology, Inc. (Lake Placid,N.Y.), New England Biolabs, Inc., (Beverly, Mass.) andCalbiochem-Novabiochem Biosciences, Inc., (La Jolla, Calif.).

Example 3 Oligonucleotide-Mediated Inhibition of J-NK1 Expression

[0170] A. JNK1 oligonucleotide sequences

[0171] Table 1 lists the nucleotide sequences of a set ofoligonucleotides designed to specifically hybridize to JNK1 mRNAs andtheir corresponding ISIS and SEQ ID numbers. The nucleotide co-ordinatesof the target gene, JNK1, and gene target regions are also included. Thenucleotide co-ordinates are derived from GenBank accession No. L26318,locus name “HUMJNK1” (see also FIG. 1(A) of Derijard et al., Cell, 1994,76, 1025). The abbreviations for gene target regions are as follows:5′-UTR, 5′ untranslated region; tIR, translation initiation region; ORF,open reading frame; 3′-UTR, 3′ untranslated region. The nucleotides ofthe oligonucleotides whose sequences are presented in Table 1 areconnected by phosphorothioate linkages and are unmodified at the 2′position (i.e., 2′-deoxy). It should be noted that the oligonucleotidetarget co-ordinate positions and gene target regions may vary withinmRNAs encoding related isoforms of JNK1 (see subsection G, below).

[0172] In addition to hybridizing to human JNK1 mRNAs, the fulloligonucleotide sequences of ISIS NOS. 12548 (SEQ ID NO: 17) and 12551(SEQ ID NO: 20) hybridize to the 5′ ends of mRNAs from Rattus norvegicus that encode a stress-activated protein kinase named “p54?”(Kyriakis et al., Nature, 1994, 369, 156).

[0173] Specifically, ISIS 12548 (SEQ ID NO: 17) hybridizes to bases498-517 of GenBank accession No. L27129, locus name “RATSAPKD,” and ISIS12551 (SEQ ID NO: 20) hybridizes to bases 803-822 of the same sequence.These oligonucleotides are thus preferred embodiments of the inventionfor investigating the role of the p54? protein kinase in rat in vitro,i.e., in cells or tissues derived from whole animals, or in vivo.

[0174] B. JNK1-specific Probes

[0175] In initial screenings of a set of oligonucleotides derived fromthe JNK1 sequence (Table 2) for biological activity, a cDNA clone ofJNK1 (Derijard et al., Cell, 1994, 76, 1025) was radiolabeled and usedas a JNK1-specific probe in Northern blots. Alternatively, however, oneor more of the oligonucleotides of Table 1 is detectably labeled andused as a JNK1-specific probe. TABLE 1 Nucleotide Sequences of JNK1Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE IDNUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES REGION 11978ATT-CTT-TCC-ACT-CTT-CTA-TT 1 1062-1081 ORF 11979CTC-CTC-CAA-GTC-CAT-AAC-TT 2 1094-1113 ORF 11980CCC-GTA-TAA-CTC-CAT-TCT-TG 3 1119-1138 ORF 11981CTG-TGC-TAA-AGG-AGA-GGG-CT 4 1142-1161 ORF 11982ATG-ATG-GAT-GCT-GAG-AGC-CA 5 1178-1197 3′-UTR 11983GTT-GAC-ATT-GAA-GAC-ACA-TC 6 1215-1234 3′-UTR 11984CTG-TAT-CAG-AGG-CCA-AAG-TC 7 1241-1260 3′-UTR 11985TGC-TGC-TTC-TAG-ACT-GCT-GT 8 1261-1280 3′-UTR 11986AGT-CAT-CTA-CAG-CAG-CCC-AG 9 1290-1309 3′-UTR 11987CCA-TCC-CTC-CCA-CCC-CCC-CA 10 1320-1339 3′-UTR 11988ATC-AAT-GAC-TAA-CCG-ACT-CC 11 1340-1359 3′-UTR 11989CAA-AAA-TAA-GAC-CAC-TGA-AT 12 1378-1397 3′-UTR 12463CAC-GCT-TGC-TTC-TGC-TCA-TG 13 0018-0037 tIR 12464CGG-CTT-AGC-TTC-TTG-ATT-GC 14 0175-0194 ORF 12538CCC-GCT-TGG-CAT-GAG-TCT-GA 15 0207-0226 ORF 12539CTC-TCT-GTA-GGC-CCG-CTT-GG 16 0218-0237 ORF 12548ATT-TGC-ATC-CAT-GAG-CTC-CA 17 0341-0360 ORF 12549CGT-TCC-TGC-AGT-CCT-GGC-CA 18 0533-0552 ORF 12550GGA-TGA-CCT-CGG-GTG-CTC-TG 19 0591-0610 ORF 12551CCC-ATA-ATG-CAC-CCC-ACA-GA 20 0646-0665 ORF 12552CGG-GTG-TTG-GAG-AGC-TTC-AT 21 0956-0975 ORF 12553TTT-GGT-GGT-GGA-GCT-TCT-GC 22 1006-1025 ORF 12554GGC-TGC-CCC-CGT-ATA-ACT-CC 23 1126-1145 ORF 12555TGC-TAA-AGG-AGA-GGG-CTG-CC 24 1139-1158 ORF 12556AGG-CCA-AAG-TCG-GAT-CTG-TT 25 1232-1251 3′-UTR 12557CCA-CCC-CCC-GAT-GGC-CCA-AG 26 1311-1330 3′-UTR

[0176] C. Activities of JNK1 Oligonucleotides

[0177] The data from screening a set of JNK1-specific phosphorothioateoligonucleotides (Table 2) indicate the following results.Oligonucleotides showing activity in this assay, as reflected by levelsof inhibition of JNK1 mRNA levels of at least 50%, include ISIS Nos.11982, 11983, 11985, 11987, 12463, 12464, 12538, 12539, 12548, 12549,12550, 12552, 12553, 12554, 12555, 12556 and 12557 (SEQ ID NOS: 5, 6, 8,10, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25 and 26,respectively). These oligonucleotides are thus preferred embodiments ofthe invention for modulating JNK1 expression. Oligonucleotides showinglevels of inhibition of JNK1 mRNAs of at least 80% in this assay,include ISIS Nos. 11982, 12539, 12464, 12548, 12554 and 12464 (SEQ IDNOS: 5, 14, 16, 17 and 23, respectively). These oligonucleotides arethus more preferred embodiments of the invention for modulating JNK1expression.

[0178] The time course of inhibition of JNK1 mRNA expression by ISIS12539 (SEQ ID NO: 16) is shown in Table 3. Following the 4 hourtreatment with ISIS 12539, the level of inhibition of JNK1 was greaterthan about 85% (t=0 h), rose to about 95% inhibition at t=4 h, andsubsequently remained at greater than or equal to about 80% (t=12 and 48h) or 60% (t=72 h). TABLE 2 Activities of JNK1 Oligonucleotides ISIS SEQID GENE TARGET % % No: NO: REGION EXPRESSION: INHIBITION: 11978 1 ORF85% 15% 11979 2 ORF 90% 10% 11980 3 ORF 85% 15% 11981 4 ORF 62% 28%11982 5 3′-UTR 13% 87% 11983 6 3′-UTR 40% 60% 11984 7 3′-UTR 53% 47%11985 8 3′-UTR 47% 53% 11986 9 3′-UTR 90% 10% 11987 10 3′-UTR 47% 53%11988 11 3′-UTR 78% 22% 11989 12 3′-UTR 60% 40% 12463 13 tIR 23% 77%12464 14 ORF 18% 82% 12538 15 ORF 33% 67% 12539 16 ORF 9% 91% 12548 17ORF 5% 95% 12549 18 ORF 28% 72% 12550 19 ORF 40% 60% 12551 20 ORF 52%48% 12552 21 ORF 34% 66% 12553 22 ORF 25% 75% 12554 23 ORF 11% 89% 1255524 ORF 27% 73% 12556 25 3′-UTR 41% 59% 12557 26 3′-UTR 29% 71%

[0179] TABLE 3 Time Course of Response to JNK1 AntisenseOligonucleotides (ASOs) SEQ ID ASO Normalized % ISIS # NO: DescriptionTime % Control Inhibition control — (LIPOFECTIN ™  0 h 100.0 0.0 only)control — (LIPOFECTIN ™  4 h 100.0 0.0 only) control — (LIPOFECTIN ™ 12h 100.0 0.0 only) control — (LIPOFECTIN ™ 48 h 100.0 0.0 only) control —(LIPOFECTIN ™ 72 h 100.0 0.0 only) 12539 16 JNK1 active  0 h 14.1 85.912539 16 ″  4 h 5.9 94.1 12539 16 ″ 12 h 11.6 88.4 12539 16 ″ 48 h 21.079.0 12539 16 ″ 272 h  41.5 58.5

[0180] D. Additional JNK1 Oligonucleotides

[0181] The results for JNK1-specific oligonucleotides (Table 2) indicatethat one of the most active phosphorothioate oligonucleotides formodulating JNK1 expression is ISIS 12539 (SEQ ID NO: 16). As detailed inTable 4, additional oligonucleotides based on this oligonucleotide weredesigned to confirm and extend the findings described above.

[0182] Oligonucleotides ISIS Nos. 14320 (SEQ ID NO: 27) and 14321 (SEQID NO: 28) are 2′-deoxy-phosphorothioate sense strand and scrambledcontrols for ISIS 12539 (SEQ ID NO: 16), respectively. ISIS Nos. 15346and 15347 are “gapmers” corresponding to ISIS 12539; both have2′-methoxyethoxy “wings” (having phosphorothioate linkages in the caseof ISIS 15346 and phosphodiester linkages in the case of ISIS 15347) anda central 2′-deoxy “gap” designed to support RNaseH activity on thetarget mRNA molecule. Similarly, ISIS Nos. 15348 to 15350 are “wingmers”corresponding to ISIS 12539 and have a 5′ or 3′ 2′-methoxyethoxyRNaseH-refractory “wing” and a 3′ or 5′ (respectively) 2′-deoxy “wing”designed to support RNaseH activity on the target JNK1 mRNA.

[0183] The chemically modified derivatives of ISIS 12539 (SEQ ID NO: 16)were tested in the Northern assay described herein at concentrations of100 and 400 nM, and the data (Table 5) indicate the following results.At 400 nM, relative to the 2′-unmodified oligonucleotide ISIS 12539,both “gapmers” (ISIS Nos. 15346 and 15347) effected inhibition of JNK1mRNA expression up to at least about 88% inhibition. Similarly, the four“wingmers” (ISIS Nos. 15348 to 15351) effected inhibition of JNK1expression of up to at least about 60 to 70% inhibition. TABLE 4Chemically Modified JNK1 Oligonucleotides SEQ ISIS NUCLEOTIDE SEQUENCE(5′ -> 3′) ID NO. AND CHEMICAL MODIFICATIONS* NO: COMMENTS 12539C^(s)T^(s)C^(s)T^(s)C^(s)T^(s)G^(s)T^(s)A^(s)G^(s)G^(s)C^(s)C^(s)C^(s)G^(s)C^(s)T^(s)T^(s)G^(s)G16 active 14320C^(s)C^(s)A^(s)A^(s)G^(s)C^(s)G^(s)G^(s)G^(s)C^(s)C^(s)T^(s)A^(s)C^(s)A^(s)G^(s)A^(s)G^(s)A^(s)G27 12539 sense control 14321C^(s)T^(s)T^(s)T^(s)C^(s)C^(s)G^(s)T^(s)T^(s)G^(s)G^(s)A^(s)C^(s)C^(s)C^(s)C^(s)T^(s)G^(s)G^(s)G28 scrambled control 15345 C ^(s) T ^(s) C ^(s) T ^(s) C ^(s) T ^(s) G^(s) T ^(s) A ^(s) G ^(s) G ^(s) C ^(s) C ^(s) C ^(s) G ^(s) C ^(s) T^(s) T ^(s) G ^(s) G 16 fully 2′- methoxyethoxy 15346 C ^(s) T ^(s) C^(s) T ^(s) C ^(s)T^(s)G^(s)T^(s)A^(s)G^(s)G^(s)C^(s)C^(s)C^(s) G ^(s) C^(s) T ^(s) T ^(s) G ^(s) G 16 “gapmer” 15347 C ^(o) T ^(o) C ^(o) T^(o) C ^(s)T^(s)G^(s)T^(s)A^(s)G^(s)G^(s)C^(s)C^(s)C^(s) G ^(o) C ^(o) T^(o) T ^(o) G ^(o) G 16 “gapmer” 15348 C ^(s) T ^(s) C ^(s) T ^(s) C^(s) T ^(s) G ^(s) T ^(s) A ^(s) G ^(s) G^(s)C^(s)C^(s)C^(s)G^(s)C^(s)T^(s)T^(s)G^(s)G 16 “wingmer” 15349C^(s)T^(s)C^(s)T^(s)C^(s)T^(s)G^(s)T^(s)A^(s) G ^(s) G ^(s) C ^(s) C^(s) C ^(s) G ^(s) C ^(s) T ^(s) T ^(s) G ^(s) G 16 “wingmer” 15351 C^(o) T ^(o) C ^(o) T ^(o) C ^(o) T ^(o) G ^(o) T ^(o) A ^(o) G ^(o) G^(s)C^(s)C^(s)C^(s)G^(s)C^(s)T^(s)T^(s)G^(s)G 16 “wingmer” 15350C^(s)T^(s)C^(s)T^(s)C^(s)T^(s)G^(s)T^(s)A^(s) G ^(o) G ^(o) C ^(o) C^(o) C ^(o) G ^(o) C ^(o) T ^(o) T ^(o) G ^(o) G 16 “wingmer” 20571 C^(s) T ^(s) C ^(s) T ^(s) C ^(s)T^(s)G^(s)T^(s)A^(s)G^(s)G^(s) C ^(s) C^(s) C ^(s) G ^(s) C ^(s) T ^(s) T ^(s) G ^(s) G 1 fully 5- methyl-cytosine version of ISIS 15346

[0184] TABLE 5 Activity of Chemically Modified JNK1 AntisenseOligonucleotides SEQ ID Oligonucleotide Normalized ISIS # NO:Description * Dose % Control control — No oligonucleotide — 100.0(LIPOFECTIN ™ only) 12539 16 JNK1 active, fully P = S & 100 nM 56.412539 16 fully 2′-deoxy 400 nM 26.7 15345 16 fully P = S & fully 2′-MOE100 nM 95.4 15345 16 400 nM 89.1 15346 16 gapmer: P = S. 2′-MOE wings;100 nM 22.6 15346 16 P = S, 2′deoxy core 400 nM 11.0 15347 16 gapmer: P= O, 2′-MOE wings; 100 nM 27.1 15347 16 P = S, 2-deoxy core 400 nM 11.715348 16 wingmer: fully P = S; 100 nM 30.4 15348 16 5′2′-MOE; 3′ 2-deoxy400 nM 32.9 15349 16 wingmer: fully P = S; 100 nM 42.5 15349 165′ 2-deoxy; 3′ 2′-MOE 400 nM 35.5 15351 16 wingmer: 5′P = O & 2′-MOE;100 nM 45.1 15351 16 3′ P = S & 2-deoxy 400 nM 39.8 15350 16 wingmer:5′ P = S & 2′- 100 nM 71.1 15350 16 deoxy; 3′ P = O & 2′-MOE 400 nM 41.3

[0185] E. Dose- and Sequence-dependent Response to JNK1 Oligonucleotides

[0186] In order to demonstrate a dose-dependent response to ISIS 12539(SEQ ID NO: 16), different concentrations (i.e., 50, 100, 200 and 400nM) of ISIS 12539 were tested for their effect on JNK1 mRNA levels inA549 cells (Table 6). In addition, two control oligonucleotides (ISIS14320, SEQ ID NO: 27, sense control, and ISIS 14321, SEQ ID NO: 28,scrambled control; see also Table 4) were also applied to A549 cells inorder to demonstrate the specificity of ISIS 12539. The results (Table6) demonstrate that the response of A549 cells to ISIS 12539 isdependent on dose in an approximately linear fashion. In contrast,neither of the control oligonucleotides effect any consistent responseon JNK1 mRNA levels.

[0187] F. Western Assays

[0188] In order to assess the effect of oligonucleotides targeted toJNK1 mRNAs on JNK1 protein levels, Western assays were performedessentially as described above in Example 2, with the followingexception(s) and/or modification(s). A primary antibody thatspecifically binds to JNK1 (catalog No. sc-474-G) was purchased fromSanta Cruz Biotechnology, Inc. (Santa Cruz, Calif.; other JNK1-specificantibodies are available from StressGen Biotechnologies, Inc., Victoria,BC, Canada; and Research Diagnostics, Inc., Flanders, N.J.). In thisexperiment, cells were grown and treated with oligonucleotide at 300 nMfor the initial 20 hours and then at 200 nM for 4 hours. At t=48 h,aliquots were removed for Northern and Western analyses, and fresh mediawas added to the cells. Aliquots for analysis were also taken at t=72 h.The samples from t=48 h and t=72 h were analyzed using the Northern andWestern assays described above. TABLE 6 Dose-Dependent Responses to JNK1Antisense Oligonucleotides SEQ ID Oligonucleotide Normalized ISIS # NO:Description Dose % Control control — No oligonucleotide — 100.0(LIPOFECTIN ™ only) 12539 16 JNK1 active  50 nM 70.3 12539 16 ″ 100 nM51.6 12539 16 ″ 200 nM 22.4 12539 16 ″ 400 nM 11.1 14320 27 12539 sensecontrol  50 nM 103.6 14320 27 ″ 100 nM 76.3 14320 27 ″ 200 nM 98.9 1432027 ″ 400 nM 97.1 14321 28 12539 scrambled control  50 nM 91.8 14321 28 ″100 nM 94.1 14321 28 ″ 200 nM 100.2 14321 28 ″ 400 nM 79.2

[0189] The data (Table 7) indicate the following results. In this assay,at t=48 h, oligonucleotides showing a level of mRNA % inhibition from >about 70% to about 100% include ISIS Nos. 12539 (phosphorothioatelinkages), 15346 and 15347 (“gapmers”), and 15348 and 15351 (5′“wingmers”) (SEQ ID NO: 16). Oligonucleotides showing levels of mRNAinhibition of from ≧ about 90% to about 100% of JNK1 mRNAs in this assayinclude ISIS Nos. 12539, 15345 AND 15346 (SEQ ID NO: 16). Theoligonucleotides tested showed approximately parallel levels of JNK1protein inhibition; ISIS Nos. 12539, 15346-15348 and 15351 effectedlevels of protein inhibition ≧ about 40%, and ISIS Nos. 12539, 15346 and15347 effected levels of protein inhibition ≧ about 55%.

[0190] At t=72 h, oligonucleotides showing a level of mRNA % inhibitionfrom > about 70% to about 100% include ISIS Nos. 12539 (phosphorothioatelinkages), 15346 and 15347 (“gapmers”), and 15348 (5′ “wingmers”) (SEQID NO: 16). Oligonucleotides showing levels of mRNA inhibition of from >about 90% to about 100% of JNK1 mRNAs at this point in the assay includeISIS Nos. 12539 and 15346 (SEQ ID NO: 16). Overall, the oligonucleotidestested showed higher levels of JNK1 protein inhibition at this point inthe assay. With the exception of the fully 2′-methoxyethoxy-modifiedISIS 15345, all of the oligonucleotides in Table 7 effect ≧ about 40%protein inhibition. ISIS Nos. 12539, 15346-15348 and 15351 effectedlevels of protein inhibition ≧ about 60%, and ISIS Nos. 12539, 15346 and15347 effected levels of protein inhibition >about 70%. TABLE 7Modulation of JNK1 mRNA and JNK1 Protein Levels by Modified JNK1Antisense Oligonucleotides SEQ ID RNA RNA % Protein Protein % ISIS # NO:% Control Inhibition % Control Inhibition t = 48 h control — 100.0 0.0100.0 0.0 12539 16 6.7 93.3 44.3 55.7 15345 16 70.3 29.7 105.0 (0.0)15346 16 4.3 95.7 42.7 57.3 15347 16 7.9 92.1 38.8 61.2 15348 16 24.375.7 58.3 41.7 15349 16 63.1 36.9 69.5 30.5 15350 16 49.2 50.8 71.7 28.315351 16 26.9 73.1 52.4 47.6 t = 72 h control 16 100.0 0.0 100.0 0.012539 16 11.7 88.3 29.2 70.8 15345 16 187.4 (0.0) 87.8 12.2 15346 1610.6 89.4 25.7 74.3 15347 16 8.2 81.8 28.4 71.6 15348 16 28.0 72.0 41.758.3 15349 16 52.0 48.0 56.5 43.5 15350 16 54.4 45.6 58.4 41.6 15351 1646.1 53.9 37.0 63.0

[0191] G. Oligonucleotides Specific for JNK1 Isoforms

[0192] Subsequent to the initial descriptions of JNK1 (Derijard et al.,Cell, 1994, 76, 1025), cDNAs encoding related isoforms of JNK1 werecloned and their nucleotide sequences determined (Gupta et al., EMBOJournal, 1996, 15, 2760). In addition to JNK1-a1 (GenBank accession No.L26318, locus name “HUMJNK1”), which encodes a polypeptide having anamino acid sequence identical to that of JNK1, the additional isoformsinclude JNK1-a2 (GenBank accession No. U34822, locus name “HSU34822”),JNK1-β1 (GenBank accession No. U35004, locus name “HSU35004”) andJNK1-β2 (GenBank accession No. U35005, locus name “HSU35005”). The fourisoforms of JNK1, which probably arise from alternative mRNA splicing,may each interact with different transcription factors or sets oftranscription factors (Gupta et al., EMBO Journal, 1996, 15, 2760). Asdetailed below, the oligonucleotides of the invention are specific forcertain members or sets of these isoforms of JNK1.

[0193] In the ORFs of mRNAs encoding JNK1/JNK1-a1 and JNK1-a2,nucleotides (nt) 631-665 of JNK1/JNK1-a1 (Genbank accession No. L26318)and nt 625-659 of JNK1-a2 (Genbank accession No. U34822) have thesequence shown below as SEQ ID NO: 63, whereas, in the ORFs of mRNAsencoding JNK1-β1 and JNK1-β2, nt 631-665 of JNK1-β1 (GenBank accessionNo. U35004) and nt 626-660 of JNK1-β2 (GenBank accession No. U35005)have the sequence shown below as SEQ ID NO: 64. For purposes ofillustration, SEQ ID NOS: 63 and 64 are shown aligned with each other(vertical marks, “|,” indicate bases that are identical in bothsequences): SEQ ID 5′-AACGTGGATTTATGGTCTGTGGGGTGCATTATGGG NO:63   ||||| ||  | ||||| || |||||||| |||||5′-AACGTTGACATTTGGTCAGTTGGGTGCATCATGGG SEQ ID NO:64

[0194] Due to this divergence between the a and b JNK1 isoforms,antisense oligonucleotides derived from the reverse complement of SEQ IDNO: 63 (i.e., SEQ ID NO: 65, see below) can be used to modulate theexpression of JNK1/JNK1-a1 and JNK1-a2 without significantly effectingthe expression of JNK1-β1 and JNK1-β2. In like fashion, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 64(i.e., SEQ ID NO: 66, see below) can be selected and used to modulatethe expression of JNK1-β1 and JNK1-β2 without significantly effectingthe expression of JNK1/JNK1-a1 and JNK1-a2. As an example, anoligonucleotide having a sequence derived from SEQ ID NO: 65 but not toSEQ ID NO: 66 is specifically hybridizable to mRNAs encodingJNK1/JNK1-a1 and JNK1-a2 but not to those encoding JNK1-β1 and JNK1-β2:SEQ ID 5′-CCCATAATGCACCCCACAGACCATAAATCCACGTT NO:65   ||||| |||||||| || |||| | | || |||||5′-CCCATGATGCACCCAACTGACCAAATGTCAACGTT SEQ ID NO:66

[0195] As a further example, in the ORFs of mRNAs encoding JNK1/JNK1-a1and JNK1-a2, nt 668-711 of JNK1/JNK1-a1 (Genbank accession No. L26318)and nt 662-705 of JNK1-a2 (Genbank accession No. U34822) have thesequence shown below as SEQ ID NO: 67, whereas, in the ORFs of mRNAsencoding JNK1-β1 and JNK1-β2, nt 668-711 of JNK1-β1 (GenBank accessionNo. U35004) and nt 663-706 of JNK1-β2 (GenBank accession No. U35005)have the sequence shown below as SEQ ID NO: 68. For purposes ofillustration, SEQ ID NOS: 67 and 68 are shown aligned with each other asfollows: 5′-AAATGGTTTGCCACAAAATCCTCTTTCCAGGAAGGGACTATATT SEQ ID NO:67   ||||| |          |  | || ||||| |  ||   |||||5′-AAATGATCAAAGGTGGTGTTTTGTTCCCAGGTACAGATCATATT SEQ ID NO:68

[0196] Due to this divergence between the a and b JNK1 isoforms,antisense oligonucleotides derived from the reverse complement of SEQ IDNO: 67 (i.e., SEQ ID NO: 69, see below) are specifically hybridizable tomRNAs encoding, and may be selected and used to modulate the expressionof, JNK1/JNK1-a1 and JNK1-a2 without significantly effecting theexpression of JNK1-β1 and JNK1-β2. In like fashion, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 68(i.e., SEQ ID NO: 70, see below) are specifically hybridizable to mRNAsencoding, and may be selected and used to modulate the expression of,can be selected and used to modulate the expression of JNK1-β1 andJNK1-β2 without significantly effecting the expression of JNK1/JNK1-a1and JNK1-a2: 5′-AATATAGTCCCTTCCTGGAAAGAGGATTTTGTGGCAAACCATTT SEQ IDNO:69    |||||  ||  | ||||| || |   |          | |||||5′-AATATGATCTGTACCTGGGAACAAAACACCACCTTTGATCATTT SEQ ID NO:70

[0197] In the case of the carboxyl terminal portion of the JNK1isoforms, JNK1/JNK1-a1 shares identity with JNK-β1; similarly, JNK1-a2and JNK1-β2 have identical carboxy terminal portions. The substantialdifferences in the amino acid sequences of these isoforms (5 amino acidsin JNK1/JNK1-a1 and JNK1-β1 are replaced with 48 amino acids in JNK1-a2and JNK1-β2) result from a slight difference in nucleotide sequence thatshifts the reading frame. Specifically, in the ORFs of mRNAs encodingJNK1/JNK1-a1 and JNK1-β1, nt 1144-1175 of JNK1/JNK1-a1 (Genbankaccession No. L26318) and JNK1-β1 (Genbank accession No. U35004) havethe sequence shown below as SEQ ID NO: 71, whereas, in the ORFs of mRNAsencoding JNK1-a2 and JNK1-β2, nt 1138-1164 of JNK1-a2 (GenBank accessionNo. U34822) and nt 1139-1165 of JNK1-β2 (GenBank accession No. U35005)have the sequence shown below as SEQ ID NO: 72. For purposes ofillustration, SEQ ID NOS: 71 and 72 are shown aligned with each other(dashes, A-,″ indicate bases that are absent in the indicated sequence,and emboldened bases indicate the stop codon for the JNK1/JNK1-a1 andJNK1-β1 ORFs): 5′-CCCTCTCCTTTAGCACAGGTGCAGCAGTGATC SEQ ID NO:71   |||||||||||||     |||||||||||||| 5′-CCCTCTCCTTTAG-----GTGCAGCAGTGATCSEQ ID NO:72

[0198] Due to this divergence between the JNK1 isoforms, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 71(i.e., SEQ ID NO: 73, see below) are specifically hybridizable to mRNAsencoding, and may be selected and used to modulate the expression of,JNK1/JNK1-a1 and JNK1-β1 without significantly effecting the expressionof JNK1-a2 and JNK1-β2. In like fashion, antisense oligonucleotidesderived from the reverse complement of SEQ ID NO: 72 (i.e., SEQ ID NO:74, see below) are specifically hybridizable to mRNAs encoding, and maybe selected and used to modulate the expression of, JNK1-a2 and JNK1-β2without significantly effecting the expression of JNK1/JNK1-a1 andJNK1-β1: 5′-GATCACTGCTGCACCTGTGCTAAAGGAGAGGG SEQ ID NO:73   ||||||||||||||     ||||||||||||| 5′-GATCACTGCTGCAC-----CTAAAGGAGAGGGSEQ ID NO:74

[0199] In preferred embodiments, such isoform-specific oligonucleotidessuch as are described above are methoxyethoxy “gapmers” or “wingmers” inwhich the RNase H-sensitive “gap” or “wing” is positioned so as tooverlap a region of nonidentity in the above antisense sequences, i.e.,SEQ ID NOS: 65, 66, 69, 70, 73 and 74.

Example 4 Oligonucleotide-Mediated Inhibition of JNK2 Expression

[0200] A. JNK2 Oligonucleotide Sequences

[0201] Table 8 lists the nucleotide sequences of oligonucleotidesdesigned to specifically hybridize to JNK2 mRNAs and the correspondingISIS and SEQ ID numbers thereof. The target gene nucleotide co-ordinatesand gene target region are also included. The nucleotide co-ordinatesare derived from GenBank accession No. L31951, locus name “HUMJNK2” (seealso FIG. 1(A) of Sluss et al., Mol. Cel. Biol., 1994, 14, 8376, andKallunki et al., Genes & Development, 1994, 8, 2996). The abbreviationsfor gene target regions are as follows: 5′-UTR, 5′ untranslated region;tIR, translation initiation region; ORF, open reading frame; 3′-UTR, 3′untranslated region. The nucleotides of the oligonucleotides whosesequences are presented in Table 8 are connected by phosphorothioatelinkages and are unmodified at the 2′ position (i.e., 2-deoxy). Itshould be noted that the oligonucleotide target co-ordinate positionsand gene target regions may vary within mRNAs encoding related isoformsof JNK2 (see subsection G, below).

[0202] In addition to hybridizing to human JNK2 mRNAs, the fulloligonucleotide sequence of ISIS No. 12562 (SEQ ID NO: 33) hybridizes tothe ORF of mRNAs from Rattus norvegicus that encode a stress-activatedprotein kinase named “p54a2” (Kyriakis et al., Nature, 1994, 369, 156).Specifically, ISIS 12562 (SEQ ID NO: 33) hybridizes to bases 649-668 ofGenBank accession No. L27112, locus name “RATSAPKB.” Thisoligonucleotide is thus a preferred embodiment of the invention forinvestigating the role of the p54a2 protein kinase in rat in vitro,i.e., in cultured cells or tissues derived from whole animals, or invivo.

[0203] B. JNK2-specific Probes

[0204] In initial screenings of a set of oligonucleotides derived fromthe JNK2 sequence (Table 9) for biological activity, a cDNA clone ofJNK2 (Kallunki et al., Genes & Development, 1994, 8, 2996) wasradiolabeled and used as a JNK2-specific probe in Northern blots.Alternatively, however, one or more of the oligonucleotides of Table 8is detectably labeled and used as a JNK2-specific probe.

[0205] C. Activities of JNK2 Oligonucleotides

[0206] The data from screening a set of JNK2-specific phosphorothioateoligonucleotides (Table 9) indicate the following results.Oligonucleotides showing activity in this assay, as reflected by levelsof inhibition of JNK2 mRNA levels of at least 50%, include ISIS Nos.12558, 12559, 12560, 12563, 12564, 12565, 12566, 12567, 12568, 12569 and12570 (SEQ ID NOS: 29, 30, 31, 34, 35, 36, 37, 38, 39, 40 and 41,respectively). These oligonucleotides are thus preferred embodiments ofthe invention for modulating JNK2 expression. Oligonucleotides showinglevels of JNK2 mRNAs of at least 80% in this assay, include ISIS Nos.12558, 12560, 12565, 12567, 12568 and 12569 (SEQ ID NOS: 29, 31, 36, 38,39 and 40, respectively). These oligonucleotides are thus more preferredembodiments of the invention for modulating JNK2 expression.

[0207] The time course of inhibition of JNK2 mRNA expression by ISIS12560 (SEQ ID NO: 31) is shown in Table 10. Following the 4 hourtreatment with ISIS 12560, the level of inhibition of JNK2 was greaterthan or equal to about 80% for at least about 12 hours and greater thanor equal to about 60% up to at least about t=48 h. TABLE 8 NucleotideSequences of JNK2 Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDESEQUENCE ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: COORDINATES REGION12558 GTT-TCA-GAT-CCC-TCG-CCC-GC 29 0003-0022 5′-UTR 12559TGC-AGC-ACA-AAC-AAT-CCC-TT 30 0168-0187 ORF 12560GTC-CGG-GCC-AGG-CCA-AAG-TC 31 0563-0582 ORF 12561CAG-GAT-GAC-TTC-GGG-CGC-CC 32 0633-0652 ORF 12562GCT-CTC-CCA-TGA-TGC-AAC-CC 33 0691-0710 ORF 12563ATG-GGT-GAC-GCA-GAG-CTT-CG 34 0997-1016 ORF 12564CTG-CTG-CAT-CTG-AAG-GCT-GA 35 1180-1199 ORF 12565TGA-GAA-GGA-GTG-GCG-TTG-CT 36 1205-1224 ORF 12566TGC-TGT-CTG-TGT-CTG-AGG-CC 37 1273-1292 ORF 12567GGT-CCC-GTC-GAG-GCA-TCA-AG 38 1295-1314 ORF 12568CAT-TTC-AGG-CCC-ACG-GAG-GT 39 1376-1395 3′-UTR 12569GGT-CTG-AAT-AGG-GCA-AGG-CA 40 1547-1566 3′-UTR 12570GGG-CAA-GTC-CAA-GCA-AGC-AT 41 1669-1688 3′-UTR

[0208] TABLE 9 Activities of JNK2 Oligonucleotides ISIS SEQ ID GENETARGET % % NO. NO: REGION EXPRESSION INHIBITION 12558 29 5′-UTR 15% 85%12559 30 ORF 28% 72% 12560 31 ORF 11% 89% 12561 32 ORF 60% 40% 12562 33ORF 89% 11% 12563 34 ORF 22% 78% 12564 35 ORF 28% 72% 12565 36 ORF 19%81% 12566 37 ORF 42% 58% 12567 38 ORF 18% 82% 12568 39 3′-UTR 20% 80%12569 40 3′-UTR 13% 87% 12570 41 3′-UTR 24% 76%

[0209] TABLE 10 Time Course of Response to JNK2 AntisenseOligonucleotides (ASOs) SEQ ID ASO Normalized % ISIS # NO: DescriptionTime % Control Inhibition control — (LIPOFECTIN ™  0 h 100.0 0.0 only)control — (LIPOFECTIN ™  4 h 100.0 0.0 only) control — (LIPOFECTIN ™ 12h 100.0 0.0 only) control — (LIPOFECTIN ™ 48 h 100.0 0.0 only) control —(LIPOFECTIN ™ 72 h 100.0 0.0 only) 12560 31 JNK2 active  0 h 20.2 79.812560 31 ″  4 h 11.1 88.9 12560 31 ″ 12 h 21.8 78.2 12560 31 ″ 48 h 42.757.3 12560 31 ″ 72 h 116.8 (0.0)

[0210] D. Additional JNK2 Oligonucleotides

[0211] The results for JNK2-specific oligonucleotides (Table 9) indicatethat one of the most active phosphorothioate oligonucleotides formodulating JNK2 expression is ISIS 12560 (SEQ ID NO: 31). As detailed inTable 11, additional oligonucleotides based on this oligonucleotide weredesigned to confirm and extend the findings described above.

[0212] Oligonucleotides ISIS Nos. 14318 (SEQ ID NO: 42) and 14319 (SEQID NO: 43) are 2′-deoxy-phosphorothioate sense strand and scrambledcontrols for ISIS 12560 (SEQ ID NO: 31), respectively. ISIS Nos. 15353and 15354 are “gapmers” corresponding to ISIS 12560; both have2′-methoxyethoxy “wings” (having phosphorothioate linkages in the caseof ISIS 15353 and phosphodiester linkages in the case of ISIS 15354) anda central 2′-deoxy “gap” designed to support RNaseH activity on thetarget mRNA molecule. Similarly, ISIS Nos. 15355 to 15358 are “wingmers”corresponding to ISIS 12560 and have a 5′ or 3′ 2′-methoxyethoxyRNaseH-refractory “wing” and a 3′ or 5′ (respectively) 2-deoxy “wing”designed to support RNaseH activity on the target JNK2 mRNA.

[0213] The chemically modified derivatives of ISIS 12560 (SEQ ID NO: 31)were tested in the Northern assay described herein at concentrations of100 and 400 nM, and the data (Table 12) indicate the following results.At 400 nM, relative to the 2′-unmodified oligonucleotide ISIS 12560,both “gapmers” (ISIS Nos. 15353 and 15354) effected approximately 80%inhibition of JNK2 mRNA expression. Similarly, the four “wingmers” (ISISNos. 15355 to 15358) effected 70-90% inhibition of JNK2 expression.

[0214] E. Dose- and Sequence-dependent Response to JNK2 Oligonucleotides

[0215] In order to demonstrate a dose-dependent response to ISIS 12560(SEQ ID NO: 31), different concentrations (i.e., 50, 100, 200 and 400nM) of ISIS 12560 were tested for their effect on JNK2 MRNA levels inA549 cells (Table 13). In addition, two control oligonucleotides (ISIS14318, SEQ ID NO: 42, sense control, and ISIS 14319, SEQ ID NO: 43,scrambled control; see also Table 11) were also applied to A549 cells inorder to demonstrate the specificity of ISIS 12560. The results (Table12) demonstrate that the response of A549 cells to ISIS 12539 isdependent on dose in an approximately linear fashion. In contrast,neither of the control oligonucleotides effect any consistent responseon JNK2 mRNA levels. TABLE 11 Chemically Modified JNK2 OligonucleotidesSEQ ISIS NUCLEOTIDE SEQUENCE (5′ -> 3′) ID NO. AND CHEMICALMODIFICATIONS* NO: COMMENTS 12560G^(S)T^(S)C^(S)C^(S)G^(S)G^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)C^(S)C^(S)A^(S)A^(S)A^(S)G^(S)T^(S)C31 active 14318G^(S)A^(S)C^(S)T^(S)T^(S)T^(S)G^(S)G^(S)C^(S)C^(S)T^(S)G^(S)G^(S)C^(S)C^(S)C^(S)G^(S)G^(S)A^(S)C42 12560 sense control 14319G^(S)T^(S)G^(S)C^(S)G^(S)C^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)C^(S)C^(S)G^(S)A^(S)A^(S)A^(S)T^(S)C43 scrambled control 15352 G ^(S) T ^(S) C ^(S) C ^(S) G ^(S) G ^(S) G^(S) C ^(S) C ^(S) A ^(S) G ^(S) G ^(S) C ^(S) C ^(S) A ^(S) A ^(S) A^(S) G ^(S) T ^(S) C 31 fully 2′- methoxyethoxy 15353 G ^(S) T ^(S) C^(S) C ^(S) G ^(S)G^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)C^(S)C^(S) A ^(S) A^(S) A ^(S) G ^(S) T ^(S) C 31 “gapmer” 15354 G ^(O) T ^(O) C ^(O) C^(O) G ^(S)G^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)C^(S)C^(S) A ^(O) A ^(O) A^(O) G ^(O) T ^(O) C 31 “gapmer” 15355 G ^(S) T ^(S) C ^(S) C ^(S) G^(S) G ^(S) G ^(S) C ^(S) C ^(S) A ^(S) G^(S)G^(S)C^(S)C^(S)A^(S)A^(S)A^(S)G^(S)T^(S)C 31 “wingmer” 15356G^(S)T^(S)C^(S)C^(S)G^(S)G^(S)G^(S)C^(S)C^(S) A ^(S) G ^(S) G ^(S) C^(S) C ^(S) A ^(S) A ^(S) A ^(S) G ^(S) T ^(S) C 31 “wingmer” 15358 G^(O) T ^(O) C ^(O) C ^(O) G ^(O) G ^(O) G ^(O) C ^(O) C ^(O) A ^(O) G^(S)G^(S)C^(S)C^(S)A^(S)A^(S)A^(S)G^(S)T^(S)C 31 “wingmer” 15357G^(S)T^(S)C^(S)C^(S)G^(S)G^(S)G^(S)C^(S)C^(S) A ^(O) G ^(O) G ^(O) C^(O) C ^(O) A ^(O) A ^(O) A ^(O) G ^(O) T ^(O) C 31 “wingmer” 20572 G^(S) T ^(S) C ^(S) C ^(S) G ^(S)G^(S)G^(S) C ^(S) C ^(S)A^(S)G^(S)G^(S)C ^(S) C ^(S) A ^(S) A ^(S) A ^(S) G ^(S) T ^(S) C 31 fully 5- methyl-cytosine version of ISIS 15353

[0216] TABLE 12 Activity of Chemically Modified JNK2 AntisenseOligonucleotides SEQ ID Oligonucleotide Normalized ISIS # NO:Description Dose % Control control — No oligonucleotide — 100.0(LIPOFECTIN ™ only) 12560 31 JNK2 active, fully P = S & 100 nM 62.112560 31 fully 2-deoxy 400 nM 31.4 15352 31 fully P = S & fully 2′-MOE100 nM 132.4 15352 31 400 nM 158.4 15353 31 gapmer: P = S, 2′-MOE 100 nM56.7 wings; 15353 31 P = S, 2-deoxy core 400 nM 21.2 15354 31 gapmer: P= O, 2′-MOE 100 nM 38.3 wings; 15354 31 P = S, 2-deoxy core 400 nM 17.115355 31 wingmer: fully P = S; 100 nM 61.3 15355 31 5′ 2′-MOE; 3′2-deoxy 400 nM 29.1 15356 31 wingmer: fully P = S; 100 nM 38.6 15356 315′ 2-deoxy; 3′ 2′-MOE 400 nM 11.0 15358 31 wingmer: 5′ P = O & 2′-MOE;100 nM 47.4 15358 31 3′ P = S & 2-deoxy 400 nM 29.4 15357 31 wingmer: 5′P = S & 2′- 100 nM 42.8 15357 31 deoxy; 3′ P = O & 2′-MOE 400 nM 13.7

[0217] TABLE 13 Dose-Dependent Responses to JNK2 AntisenseOligonucleotides SEQ ID Oligonucleotide Normalized ISIS # NO:Description Dose % Control control — No oligonucleotide — 100.0(LIPOFECTIN ™ only) 12560 31 JNK2 active  50 nM 68.1 12560 31 ″ 100 nM50.0 12560 31 ″ 200 nM 25.1 12560 31 ″ 400 nM 14.2 14318 42 12560 sensecontrol  50 nM 87.1 14318 42 ″ 100 nM 89.8 14318 42 ″ 200 nM 92.1 1431842 ″ 400 nM 99.6 14319 43 12560 scrambled control  50 nM 90.4 14319 43 ″100 nM 93.7 14319 43 ″ 200 nM 110.2 14319 43 ″ 400 nM 100.0

[0218] F. Western Assays

[0219] In order to assess the effect of oligonucleotides targeted toJNK2 mRNAs on JNK2 protein levels, Western assays are performedessentially as described above in Examples 2 and 3. A primary antibodythat specifically binds to JNK2 is purchased from, for example, SantaCruz Biotechnology, Inc., Santa Cruz, Calif.; Upstate Biotechnology,Inc., Lake Placid, N.Y.; StressGen Biotechnologies, Inc., Victoria, BC,Canada; or Research Diagnostics, Inc., Flanders, N.J.

[0220] G. Oligonucleotides Specific for JNK2 Isoforms

[0221] Subsequent to the initial descriptions of JNK2 (Sluss et al.,Mol. Cel. Biol., 1994, 14, 8376; Kallunki et al., Genes & Development,1994, 8, 2996; GenBank accession No. HSU09759, locus name “U09759”),cDNAs encoding related isoforms of JNK2 were cloned and their nucleotidesequences determined (Gupta et al., EMBO Journal, 1996, 15, 2760). Inaddition to JNK2-a2 (GenBank accession No. L31951, locus name“HUMJNK2”), which encodes a polypeptide having an amino acid sequenceidentical to that of JNK2, the additional isoforms include JNK2-a1(GenBank accession No. U34821, locus name “HSU34821”), JNK2-β1 (GenBankaccession No. U35002, locus name “HSU35002”) and JNK2-β2 (GenBankaccession No. U35003, locus name “HSU35003”). The four isoforms of JNK2,which probably arise from alternative mRNA splicing, may each interactwith different transcription factors or sets of transcription factors(Gupta et al., EMBO Journal, 1996, 15, 2760). As detailed below, theoligonucleotides of the invention are specific for certain members orsets of these isoforms of JNK2.

[0222] In the ORFs of mRNAs encoding JNK2/JNK2-a2 and JNK2-a1,nucleotides (nt) 689-748 of JNK2/JNK2-a2 (GenBank accession No. L31951)and nt 675-734 of JNK2-a1 (GenBank accession No. U34821) have thesequence shown below as SEQ ID NO: 75, whereas, in the ORFs of mRNAsencoding JNK2-β1 and JNK2-β2, nt 653-712 of JNK2-β1 (GenBank accessionNo. U35002) and nt 665-724 of JNK2-β2 (GenBank accession No. U35003)have the sequence shown below as SEQ ID NO: 76. For purposes ofillustration, SEQ ID NOS: 75 and 76 are shown aligned with each other(vertical marks, “|,” indicate bases that are identical in bothsequences):5′-GTGGGTTGCATCATGGGAGAGCTGGTGAAAGGTTGTGTGATATTCCAAGGCACTGACCAT SEQ IDNO:75    || || |||||||||| |||  ||||      |   ||  | ||||  || |  ||| ||5′-GTCGGGTGCATCATGGCAGAAATGGTCCTCCATAAAGTCCTGTTCCCGGGAAGAGACTAT

[0223] SEQ ID NO: 76

[0224] Due to this divergence between the a and b JNK2 isoforms,antisense oligonucleotides derived from the reverse complement of SEQ IDNO: 75 (i.e., SEQ ID NO: 77, see below) are specifically hybridizableto, and may be selected and used to modulate the expression of,JNK2/JNK2-a2 and JNK2-a1 without significantly effecting the expressionof JNK1-β1 and JNK1-β2. In like fashion, antisense oligonucleotidesderived from the reverse complement of SEQ ID NO: 76 (i.e., SEQ ID NO:78, see below) are specifically hybridizable to, and may be selected andused to modulate the expression of, JNK2-β1 and JNK2-β2 withoutsignificantly effecting the expression of JNK2/JNK2-a2 and JNK2-a1. Asan example, an oligonucleotide having a sequence derived from SEQ ID NO:77 but not from SEQ ID NO: 78 is specifically hybridizable to, mRNAsencoding JNK1/JNK1-a1 and JNK1-a2 but not to those encoding JNK2-β1 andJNK2-β2: 5′-ATGGTCAGTGCCTTGGAATATCACACAACCTTTCACCAGCTCTCCCATGATGCAACCCACSEQ ID NO:77   || |||  | ||  |||| |  ||   |      ||||  ||| |||||||||| || ||5′-ATAGTCTCTTCCCGGGAACAGGACTTTATGGAGGACCATTTCTGCCATGATGCACCCGAC

[0225] SEQ ID NO: 78

[0226] In the case of the carboxyl terminal portion of the JNK2isoforms, JNK2/JNK2-a2 shares identity with JNK1-β2; similarly, JNK2-a1and JNK2-β1 have identical carboxy terminal portions. The substantialdifferences in the amino acid sequences of these isoforms (5 amino acidsin JNK2-a2 and JNK2-β2 are replaced with 47 amino acids in JNK2/JNK2-a2and JNK2-β2) result from a slight difference in nucleotide sequence thatshifts the reading frame. Specifically, in the ORFs of mRNAs encodingJNK2-a1 and JNK1-β1, nt 1164-1198 of JNK2-a1 (GenBank accession No.U34821) and nt 1142-1176 of JNK2-β1 (GenBank accession No. U35002) havethe sequence shown below as SEQ ID NO: 79, whereas, in the ORFs of mRNAsencoding JNK2/JNK2-a2 and JNK2-β2, nt 1178-1207 of JNK2/JNK2-a2 (GenBankaccession No. L31951) and nt 1154-1183 of JNK2-β2 (GenBank accession No.U35003) have the sequence shown below as SEQ ID NO: 80. For purposes ofillustration, SEQ ID NOS: 79 and 80 are shown aligned with each other(dashes, “-,” indicate bases that are absent in the indicated sequence,and emboldened bases indicate the stop codon for the JNK2-a1 and JNK2-β1ORFs): SEQ ID 5′-GATCAGCCTTCAGCACAGATGCAGCAGTAAGTAGC NO:79   |||||||||||||     |||||||||||||||||5′-GATCAGCCTTCAG-----ATGCAGCAGTAAGTAGC SEQ ID NO:80

[0227] Due to this divergence between the JNK2 isoforms, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 79(i.e., SEQ ID NO: 81, see below) are specifically hybridizable to, andmay be selected and used to modulate the expression of, mRNAs encodingJNK2-a1 and JNK2-β1 without significantly effecting the expression ofJNK2/JNK2-a2 and JNK2-β2. In like fashion, antisense oligonucleotidesderived from the reverse complement of SEQ ID NO: 80 (i.e., SEQ ID NO:82, see below) are specifically hybridizable to, and may be selected andused to modulate the expression of, mRNAs encoding JNK2/JNK2-a2 andJNK2-β2 without significantly effecting the expression of JNK2-a1 andJNK2-β1. As an example, ISIS 12564 (SEQ ID NO: 35) corresponds to SEQ IDNO: 82 but not to SEQ ID NO: 81, and is thus specifically hybridizableto, and may be used to modulate the expression of, mRNAs encodingJNK2/JNK2-a2 and JNK2-β2 but not those encoding JNK2-a1 and JNK2-a1:5′-GCTACTTACTGCTGCATCTGTGCTGAAGGCTGATC SEQ ID NO:81   |||||||||||||||||     ||||||||||||5′-GCTACTTACTGCTGCAT-----CTGAAGGCTGATC SEQ ID NO:82           |||||||||     |||||||||||        5′-CTGCTGCAT-----CTGAAGGCTGA SEQ ID NO:35

[0228] In preferred embodiments, such isoform-specific oligonucleotidessuch as are described above are methoxyethoxy “gapmers” or “wingmers” inwhich the RNase H-sensitive “gap” or “wing” is positioned so as tooverlap a region of nonidentity in the above antisense sequences, i.e.,SEQ ID NOS: 77, 78, 81 and 82.

Example 5 Oligonucleotide-Mediated Inhibition of JNK3 Expression

[0229] A. JNK3 Oligonucleotide Sequences

[0230] Table 14 lists the nucleotide sequences of oligonucleotidesdesigned to specifically hybridize to JNK3 mRNAs and the correspondingISIS and SEQ ID numbers thereof. The target gene nucleotide co-ordinatesand gene target region are also included. The nucleotide co-ordinatesare derived from GenBank accession No. U07620, locus name “HSU07620” seealso FIG. 4(A) of Mohit et al., Neuron, 1994, 14, 67). The abbreviationsfor gene target regions are as follows: 5′-UTR, 5′ untranslated region;tIR, translation initiation region; ORF, open reading frame; 3′-UTR, 3′untranslated region. It should be noted that the oligonucleotide targetco-ordinate positions and gene target regions may vary within mRNAsencoding related isoforms of JNK3 (see subsection D, below).

[0231] The nucleotides of the oligonucleotides whose sequences arepresented in Table 14 are connected by phosphorothioate linkages and are“gapmers.” Specifically, the six nucleotides of the 3′ and 5′ terminiare 2′-methoxyethoxy- modified and are shown emboldened in Table 14,whereas the central eight nucleotides are unmodified at the 2′ position(i.e., 2-deoxy).

[0232] In addition to hybridizing to human JNK3 mRNAs, the fulloligonucleotide sequences of ISIS Nos. 16692, 16693, 16703, 16704,16705, 16707, and 16708 (SEQ ID NOS: 46, 47, 56, 57, 58, 60 and 61,respectively) specifically hybridize to mRNAs from Rattus norvegicusthat encode a stress-activated protein kinase named “p54β” (Kyriakis etal., Nature, 1994, 369, 156; GenBank accession No. L27128, locus name“RATSAPKC.” Furthermore, the full oligonucleotide sequences of 16692,16693, 16695, 16703, 16704, 16705, 16707 and 16708 (SEQ ID NOS: 46, 47,49, 56, 57, 58, 60 and 61, respectively) specifically hybridize to mRNAsfrom Mus musculus that encode a mitogen activated protein (MAP) kinasestress activated protein named the “p459^(3F12) SAP kinase” (Martin etal., Brain Res. Mol. Brain Res., 1996, 35, 47; GenBank accession No.L35236, locus name “MUSMAPK”). These oligonucleotides are thus preferredembodiments of the invention for investigating the role of the p54β andp459^(3F12) SAP protein kinases in rat or mouse, respectively, in vitro,i.e., in cultured cells or tissues derived from whole animals or invivo. The target gene nucleotide co-ordinates and gene target regionsfor these oligonucleotides, as defined for these GenBank entries, aredetailed in Table 15.

[0233] B. JNK3-specific Probes

[0234] In initial screenings of a set of oligonucleotides derived fromthe JNK3 sequence for biological activity, a cDNA clone of JNK3(Derijard et al., Cell, 1994, 76, 1025) was radiolabeled and used as aJNK3-specific probe in Northern blots. Alternatively, however, one ormore of the oligonucleotides of Table 14 is detectably labeled and usedas a JNK3-specific probe.

[0235] C. Western Assays

[0236] In order to assess the effect of oligonucleotides targeted toJNK3 mRNAs on JNK3 protein levels, Western assays are performedessentially as described above in Examples 2 through 4. A primaryantibody that specifically binds to JNK3 is purchased from, for example,Upstate Biotechnology, Inc. (Lake Placid, N.Y.), StressGenBiotechnologies Corp. (Victoria, BC, Canada), or New England Biolabs,Inc. (Beverly, Mass.). TABLE 14 Nucleotide Sequences of JNK3Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ IDNUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES REGION 16690TTC-AAC-AGT-TTC-TTG-CAT-AA 44 0157-0176 5′-UTR 16691CTC-ATC-TAT-AGG-AAA-CGG-GT 45 0182-0200 5′-UTR 16692TGG-AGG-CTC-ATA-AAT-ACC-AC 46 0215-0234 tIR 16693TAT-AAG-AAA-TGG-AGG-CTC-AT 47 0224-0243 tIR 16694TCA-CAT-CCA-ATG-TTG-GTT-CA 48 0253-0272 ORF 16695TTA-TCG-AAT-CCC-TGA-CAA-AA 49 0281-0300 ORF 16696GTT-TGG-CAA-TAT-ATG-ACA-CA 50 0310-0329 ORF 16697CTG-TCA-AGG-ACA-GCA-TCA-TA 51 0467-0486 ORF 16698AAT-CAC-TTG-ACA-TAA-GTT-GG 52 0675-0694 ORF 16699TAA-ATC-CCT-GTG-AAT-AAT-TC 53 0774-0793 ORF 16700GCA-TCC-CAC-AGA-CCA-TAT-AT 54 0957-0976 ORF 16702TGT-TCT-CTT-TCA-TCC-AAC-TG 55 1358-1377 ORF 16703TCT-CAC-TGC-TGT-TCA-CTG-CT 56 1485-1504 tIR 16704GGG-TCT-GGT-CGG-TGG-ACA-TG 57 1542-1561 3′-UTR 16705AGG-CTG-CTG-TCA-GTG-TCA-GA 58 1567-1586 3′-UTR 16706TCA-CCT-GCA-ACA-ACC-CAG-GG 59 1604-1623 3′-UTR 16707GCG-GCT-AGT-CAC-CTG-CAA-CA 60 1612-1631 3′-UTR 16708CGC-TGG-GTT-TCG-CAG-GCA-GG 61 1631-1650 3′-UTR 16709ATC-ATC-TCC-TGA-AGA-ACG-CT 62 1647-1666 3′-UTR

[0237] TABLE 15 Rat and Mouse Gene Target Locations of JNK3Oligonucleotides Mouse Rat p54β p459^(3F12) SEQ NUCLEOTIDE GENENUCLEOTIDE GENE ISIS ID CO- TARGET CO- TARGET NO. NO: ORDINATES¹ REGIONORDINATES² REGION 16692 46 0213-0232 5′-UTR 0301-0320 tIR 16693 470222-0241 5′-UTR 0310-0329 tIR 16695 49 — — 0367-0386 ORF 16703 561506-1525 ORF 1571-1590 tTR 16704 57 1563-1582 ORF 1628-1647 3′-UTR16705 58 1588-1607 ORF 1653-1672 3′-UTR 16707 60 1633-1652 tTR 1698-17173′-UTR 16708 61 1652-1671 3′-UTR 1717-1736 3′-UTR

[0238] D. Oligonucleotides Specific for JNK3 Isoforms

[0239] Two isoforms of JNK3 have been described. JNK3-a1 was initiallycloned and named “p₄₉ ^(3F12) kinase” by (Mohit et al. Neuron, 1995, 14,67). Subsequently, two cDNAs encoding related isoforms of JNK3 werecloned and their nucleotide sequences determined (Gupta et al., EMBOJournal, 1996, 15, 2760). The isoforms are named JNK3-a1 (GenBankaccession No. U34820, locus name “HSU34820”) and JNK3-a2 (GenBankaccession No. U34819, locus name “HSU34819”) herein. The two isoforms ofJNK3, which probably arise from alternative mRNA splicing, may eachinteract with different transcription factors or sets of transcriptionfactors (Gupta et al., EMBO Journal, 1996, 15, 2760). As detailed below,certain oligonucleotides of the invention are specific for each of theseisoforms of JNK3.

[0240] JNK3-a1 and JNK-a2 differ at their carboxyl terminal portions.The substantial differences in the amino acid sequences of theseisoforms (5 amino acids in JNK3-a1 are replaced with 47 amino acids inJNK3-a2) result from a slight difference in nucleotide sequence thatshifts the reading frame. Specifically, in the ORF of mRNAs encodingJNK3-a1, nucleotides (nt) 1325-1362 of JNK3-a1 (GenBank accession No.U34820) have the sequence shown below as SEQ ID NO: 83, whereas, in theORF of mRNAs encoding JNK3-a2, nt 1301-1333 of JNK3-a2 (GenBankaccession No. U34819) have the sequence shown below as SEQ ID NO: 84.For purposes of illustration, SEQ ID NOS: 83 and 202 are shown alignedwith each other (vertical marks, “|,” indicate bases that are identicalin both sequences; dashes, “-,” indicate bases that are absent in theindicated sequence; and emboldened bases indicate the stop codon for theJNK3-a1 ORF): SEQ ID 5′-GGACAGCCTTCTCCTTCAGCACAGGTGCAGCAGTGAAC NO:83   |||||||||||||||||||     ||||||||||||||5′-GGACAGCCTTCTCCTTCAG-----GTGCAGCAGTGAAC SEQ ID NO:84

[0241] Due to this divergence between the JNK3 isoforms, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 83(i.e., SEQ ID NO: 85, see below) are specifically hybridizable to mRNAsencoding JNK3-a1, and may be selected and used to modulate theexpression of JNK3-a1 without significantly effecting the expression ofJNK3-a2. In like fashion, antisense oligonucleotides derived from thereverse complement of SEQ ID NO: 84 (i.e., SEQ ID NO: 86, see below) arespecifically hybridizable to mRNAs encoding JNK3-a2, and may be selectedand used to modulate the expression of JNK3-a2 without significantlyeffecting the expression of JNK3-a1: SEQ ID5′-GTTCACTGCTGCACCTGTGCTGAAGGAGAAGGCTGTCC NO:85   ||||||||||||||     |||||||||||||||||||5′-GTTCACTGCTGCAC-----CTGAAGGAGAAGGCTGTCC SEQ ID NO:86

[0242] In preferred embodiments, such isoform-specific oligonucleotidessuch as are described above are methoxyethoxy “gapmers” or “wingmers” inwhich the RNase H-sensitive “gap” or “wing” is positioned so as tooverlap a region of nonidentity in the above antisense sequences, i.e.,SEQ ID NOS: 85 and 86.

[0243] E. Activities of JMM3 Oligonucleotides

[0244] The JNK3-specific phosphorothioate, 2′-methoxyethoxy “gapmer”oligonucleotides (Table 14) were screened for their ability to affectJNK3 mRNA levels in SH-SY5Y cells (Biedler et al., Cancer Res., 1973,33, 2643). SH-SY5Y cells express a variety of mitogen-activated proteinkinases (MAPKs; see, e.g., Cheng et al., J. Biol. Chem., 1998, 273,14560). Cells were grown in DMEM essentially as previously described(e.g., Singleton et al., J. Biol. Chem., 1996, 271, 31791; Jalava etal., Cancer Res., 1990, 50, 3422) and treated with oligonucleotides at aconcentration of 200 nM as described in Example 2. Control cultures weretreated with an aliquot of LIPOFECTIN™ that contained nooligonucleotide.

[0245] The results are shown in Table 16. Oligonucleotides showinglevels of inhibition of JNK3 mRNA levels of at least 45% include ISISNos. 16692, 16693, 16694, 16695, 16696, 16697, 16702, 16703, 16704,16705 and 16706 (SEQ ID NOS:46, 47, 48, 49, 50, 51, 55, 56, 57, 58 and59, respectively). These oligonucleotides are preferred embodiments ofthe invention for modulating JNK3 expression. Oligonucleotidesinhibiting JNK3 mRNAs by at least 60% in this assay include ISIS Nos.16693, 16702, 16703 and 16704 (SEQ ID NOS: 47, 55, 56 and 57,respectively). These oligonucleotides are thus more preferredembodiments of the invention for modulating JNK3 expression. TABLE 16Activities of JNK3 Oligonucleotides SEQ ISIS ID GENE TARGET % % No: NO:REGION EXPRESSION: INHIBITION: ¹control — — 100%   0% 16690 44 5′-UTR60% 40% 16691 45 5′-UTR 66% 34% 16692 46 tIR 47% 53% 16693 47 tIR 40%60% 16694 48 ORF 42% 58% 16695 49 ORF 44% 56% 16696 50 ORF 55% 45% 1669751 ORF 54% 46% 16698 52 ORF 63% 37% 16699 53 ORF 61% 39% 16700 54 ORFN.D.² N.D. 16702 55 ORF 39% 61% 16703 56 tTR 30% 70% 16704 57 3′-UTR 36%64% 16705 58 3′-UTR 42% 58% 16706 59 3′-UTR 45% 55% 16707 60 3′-UTR 73%27% 16708 61 3′-UTR 68% 32% 16709 62 3′-UTR 66% 34%

Example 6 Effect of Oligonucleotides Targeted to AP-1 Subunits onEnzymes Involved in Metastasis

[0246] Patients having benign tumors, and primary malignant tumors thathave been detected early in the course of their development, may oftenbe successfully treated by the surgical removal of the benign or primarytumor. If unchecked, however, cells from malignant tumors are spreadthroughout a patient's body through the processes of invasion andmetastasis. Invasion refers to the ability of cancer cells to detachfrom a primary site of attachment and penetrate, e.g., an underlyingbasement membrane. Metastasis indicates a sequence of events wherein (1)a cancer cell detaches from its extracellular matrices, (2) the detachedcancer cell migrates to another portion of the patient's body, often viathe circulatory system, and (3) attaches to a distal and inappropriateextracellular matrix, thereby created a focus from which a secondarytumor can arise. Normal cells do not possess the ability to invade ormetastasize and/or undergo apoptosis (programmed cell death) if suchevents occur (Ruoslahti, Sci. Amer., 1996, 275, 72).

[0247] The matrix metalloproteinases (MMPs) are a family of enzymeswhich have the ability to degrade components of the extracellular matrix(Birkedal-Hansen, Current Op. Biol., 1995, 7, 728). Many members of theMMP family have been found to have elevated levels of activity in humantumors as well as other disease states (Stetler-Stevenson et al., Annu.Rev. Cell Biol., 1993, 9, 541; Bernhard et al., Proc. Natl. Acad. Sci.(U.S.A.), 1994, 91, 4293). In particular, one member of this family,matrix metalloproteinase-9 (MMP-9), is often found to be expressed onlyin tumors and other diseased tissues (Himelstein et al., Invasion &Metastasis, 1994, 14, 246). Several studies have shown that regulationof the MMP-9 gene may be controlled by the AP-1 transcription factor(Kerr et al., Science, 1988, 242, 1242; Kerr et al., Cell, 1990, 61,267; Gum et al., J. Biol. Chem., 1996, 271, 10672; Hua et al., CancerRes., 1996, 56, 5279). In order to determine whether MMP-9 expressioncan be influenced by AP-1 modulation, the following experiments wereconducted on normal human epidermal keratinocytes (NHEKs). AlthoughNHEKs normally express no detectable MMP-9, MMP-9 can be induced by anumber of stimuli, including TPA (12-O-tetradecanoylphorbol 13-acetate).ISIS 10582, an oligonucleotide targeted to c-jun, was evaluated for itsability to modulate MMP-9 expression (see pending application Ser. No.08/837,201, filed Apr. 14, 1997, attorney docket No. ISPH-0209. Theresults (Table 16) demonstrate that ISIS 10582 is able to completelyinhibit the expression of MMP-9 after induction with TPA. TABLE 17Effect of c-jun Oligonucleotide on MMP-9 Expression Treatment MMP-9Basal 4 TPA-no oligo 100 10582: c-jun active 6 11562: sense control 9911563: scrambled control 95 11564: mismatch control 89

[0248] These results demonstrate that c-Jun is required for TPA-mediatedinduction of MMP-9, and indicate that oligonucleotides targeted to AP-1subunits can inhibit the expression of MMP family members, therebymodulating the ability of cancer cells to invade other tissues and/ormetastasize to other sites in a patient's body. Because JNK proteinsactivate AP-1 by phosphorylating the N-terminal portion of the Junsubunit thereof, modulation of one or more JNK proteins by theoligonucleotides of the present disclosure will also modulate theexpression of MMP family members and limit the metastatic ability ofcancer cells.

Example 7 Treatment of Human Tumors in Mice with OligonucleotidesTargeted to JNK Proteins

[0249] Approximately 5×10⁶ breast adenocarcinoma cells (cell lineMDA-MB-231; American Type Culture Collection, Richmond, Va., No. ATCCHTB-26) were implanted subcutaneously in the right inner thigh of nudemice (n=6 for each of three sets of mice). Oligonucleotides ISIS 15346(JNK1, SEQ ID NO:16) and 15353 (JNK2, SEQ ID NO:31) were suspended insaline and administered once daily to two sets of mice on the first daythe tumor volume was about 100 mm³. A saline-only (0.9% NaCl) solutionwas given to a third set of animals as a control. Oligonucleotides weregiven by intravenous injection at a dosage of 25 mg/kg. Tumor size wasmeasured and tumor volume was calculated on days 12, 19, 26 and 33following tumor cell inoculation.

[0250] The results are shown in Table 18. Both 15346 (JNK1, SEQ IDNO:16) and 15353 (JNK2, SEQ ID NO:31) inhibited tumor growth compared tothe saline control. Specifically, on days 26 and 33, the MDA-MB-231tumors in animals that had been treated with the oligonucleotides hadsmaller volumes than the tumors in saline-treated animals, indicatingthat the oligonucleotides inhibited the growth of the tumors.

[0251] The antisense compounds of the invention are also tested fortheir ability to slow or eliminate the growth of xenografts resultingfrom, for example, human cervical epithelial carcinoma cells (HeLa cellline, ATCC No. ATCC CCL-2), human lung carcinoma cells (cell line A549,ATCC No. ATCC CCL-185), human adenocarcinoma cells (cell line SW480,ATCC No. ATCC CCL-228), human bladder carcinoma cells (cell line T24,ATCC No. HTB-4), human pancreatic carcinoma cells (cell line MIA PaCa,ATCC No. CRL-1420) and human small cell carcinoma cells (cell lineNCI-H69, ATCC HTB-119). Xenografts resulting from these and other celllines are established using essentially the same techniques as were usedfor the experiments using MDA-MB 231 cells. TABLE 18 Response ofMDA-MB-231 Tumors in Mice to Oligonucleotides Targeted to JNK1 and JNK2Mean Tumor Standard Standard Treatment: Volume (cm³) Deviation ErrorSaline: Day 12 0.122 0.053 0.022 Day 19 0.253 0.078 0.032 Day 26 0.6480.265 0.108 Day 33 1.560 0.887 0.362 ISIS (JNK1): Day 12 0.122 0.0330.014 Day 19 0.255 0.099 0.040 Day 26 0.400 0.202 0.083 Day 33 0.6380.416 0.170 ISIS (JNK2): Day 12 0.122 0.041 0.017 Day 19 0.230 0.0720.029 Day 26 0.358 0.131 0.053 Day 33 0.762 0.366 0.150

Example 8 Oligonucleotides Targeted to Genes Encoding Rat JNK Proteins

[0252] In order to study the role of JNK proteins in animal models,oligonucleotides targeted to the genes encoding JNK1, JNK2 and JNK3 ofRattus norvegicus were prepared. These oligonucleotides are2′-methoxyethoxy, phosphodiester/2′-hydroxyl,phosphorothioate/2′-methoxyethoxy, phosphodiester “gapmers” in whichevery cytosine residue is 5-methylcytosine (msc). These antisensecompounds were synthesized according to the methods of the disclosure.Certain of these oligonucleotides are additionally specificallyhybridizable to JNK genes from other species as indicated herein. Theoligonucleotides described in this Example were tested for their abilityto modulate rat JNK mRNA levels essentially according to the methodsdescribed in the preceding Examples, with the exceptions that the cellline used was rat A10 aortic smooth muscle cells (ATCC No. ATCCCRL-1476) and the probes used were specific for rat JNK1, JNK2 or JNK3(see infra). A10 cells were grown and treated with oligonucleotidesessentially as described by (Cioffi et al. Mol. Pharmacol., 1997, 51,383).

[0253] A. JNK1: Table 19 describes the sequences and structures of a setof oligonucleotides, ISIS Nos. 21857 to 21870 (SEQ ID NOS:111 to 124,respectively) that were designed to be specifically hybridizable tonucleic acids from Rattus norvegicus that encode a stress-activatedprotein kinase named “p54?” or “SAPK?” that is homologous to the humanprotein JNK1 (Kyriakis et al., Nature, 1994, 369, 156; GenBank accessionNo. L27129, locus name “RATSAPKD”). In Table 19, emboldened residues are2′-methoxyethoxy-residues (others are 2′-deoxy-); “C” residues are2′-methoxyethoxy-5-methyl-cytosines and “C” residues are5-methyl-cytosines; “o” indicates a phosphodiester linkage; and “s”indicates a phosphorothioate linkage. The target gene co-ordinates arefrom GenBank Accession No. L27129, locus name “RATSAPKD.” TABLE 19Nucleotide Sequences of Rat JNK1 Oligonucleotides SEQ TARGET GENE GENEISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO COORDREGION 21857 CoAoAoCoGsTsCsCsCsGsCsGsCsTsCsGoGoCoCoG 111 0002-00215′-UTR 21858 CoCoToGoCsTsCsGsCsGsGsCsTsCsCsGoCoGoToT 112 0029-00485′-UTR 21859 CoToCoAoTsGsAsTsGsGsCsAsAsGsCsAoToToAoA 113 0161-0180 tIR21860 ToGoToToGsTsCsAsCsGsTsTsTsAsCsToToCoToG 114 0181-0200 ORF 21861CoGoGoToAsGsGsCsTsCsGsCsTsTsAsGoCoAoToG 115 0371-0390 ORF 21862CoToAoGoGsGsAsTsTsTsCsTsGsTsGsGoToGoToG 116 0451-0470 ORF 21863CoAoGoCoAsGsAsGsTsGsAsAsGsGsTsGoCoToToG 117 0592-0611 ORF 21864ToCoGoToTsCsCsTsGsCsAsGsTsCsCsToToGoCoC 118 0691-0710 ORF 21865CoCoAoToTsTsCsTsCsCsCsAsTsAsAsToGoCoAoC 119 0811-0830 ORF 21866ToGoAoAoTsTsCsAsGsGsAsCsAsAsGsGoToGoToT 120 0901-0920 ORF 21867AoGoCoToTsCsGsTsCsTsAsCsGsGsAsGoAoToCoC 121 1101-1120 ORF 21868CoAoCoToCsCsTsCsTsAsTsTsGsTsGsToGoCoToC 122 1211-1230 ORF 21869GoCoToGoCsAsCsCsTsAsAsAsGsGsAsGoAoCoGoG 123 1301-1320 ORF 21870CoCoAoGoAsGsTsCsGsGsAsTsCsTsGsToGoGoAoC 124 1381-1400 ORF

[0254] These antisense compounds were tested for their ability tomodulate levels of p54? (JNK1) and p54a (JNK2) mRNA in A10 cells viaNorthern assays. Due to the high degree of sequence identity between thehuman and rat genes, radiolabeled human JNK1 (Example 3) and JNK2(Example 4) cDNAs functioned as specific probes for the rat homologs.

[0255] The results are shown in Table 20. ISIS Nos. 21857 to 21870 (SEQID NOS:111 to 124, respectively) showed 70% to 90% inhibition of ratJNK1 mRNA levels. These oligonucleotides are preferred embodiments ofthe invention for modulating rat JNK1 expression. oligonucleotidesshowing levels of inhibition of at least 90% in this assay include ISISNos. 21858, 21859, 21860, 21861, 21862, 21864, 21865, 21866 and 21867(SEQ ID NOS:112, 113, 114, 115, 116, 118, 119, 120 and 121,respectively). These oligonucleotides are thus more preferredembodiments of the invention for modulating rat JNK1 expression. ISIS21859 (SEQ ID NO:113) was chosen for use in further studies (infra).

[0256] Two of the oligonucleotides, ISIS Nos. 21861 and 21867 (SEQ IDNOS:115 and 121, respectively) demonstrated a capacity to modulate bothJNK1 and JNK2. Such oligonucleotides are referred to herein as “Pan JNK”antisense compounds because the term “Pan” is used in immunologicalliterature to refer to an antibody that recognizes, e.g., all isoformsof a protein or subtypes of a cell type. The Pan JNK oligonucleotidesare discussed in more detail infra.

[0257] In addition to being specifically hybridizable to nucleic acidsencoding rat JNK1, some of the oligonucleotides described in Table R-1are also specifically hybridizable with JNK1-encoding nucleic acids fromother species. ISIS 21859 (SEQ ID NO:113) is complementary to bases 4 to23 of cDNAs encoding human JNK1a1 and JNK1β1 (i.e., GenBank accessionNos. L26318 and U35004, respectively). ISIS 21862 (SEQ ID NO:116) iscomplementary to bases 294 to 313 of the human JNK1a1 and JNK1β1 cDNAs(GenBank accession Nos. L26318 and U35004, respectively), bases 289 to308 of the human JNK1β2 cDNA (GenBank accession No. U35005), and bases288 to 307 of the human JNK1a2 cDNA (GenBank accession No. U34822).Finally, ISIS 21865 is complementary to bases 654 to 673 of the humanJNK1a1 cDNA (GenBank accession No. L26318) and to bases 648 to 667 ofthe human JNK1a2 cDNA (GenBank accession No. U34822). Theseoligonucleotides are tested for their ability to modulate mRNA levels ofhuman JNK1 genes according to the methods described in Example 3. TABLE20 Activities of Oligonucleotides Targeted to Rat JNK1 SEQ ISIS ID GENETARGET % EXPRESSION % EXPRESSION No: NO: REGION JNK1 JNK2 control¹ — —100% 100% 21857 111 5′-UTR 24% 91% 21858 112 5′-UTR 8% 89% 21859 113 tIR5% 106% 21860 114 ORF 8% 98% 21861 115 ORF 6% 13% 21862 116 ORF 6% 133%21863 117 ORF 24% 107% 21864 118 ORF 8% 106% 21865 119 ORF 5% 50% 21866120 ORF 8% 98% 21867 121 ORF 5% 21% 21868 122 ORF 15% 112% 21869 123 ORF30% 93% 21870 124 ORF 11% 87%

[0258] B. JNK2

[0259] Table 21 describes the sequences and structures of a set ofoligonucleotides, ISIS Nos. 18254 to 18267 (SEQ ID NOS:125 to 138,respectively) that were designed to be specifically hybridizable tonucleic acids that encode a stress-activated protein kinase from Rattusnorvegicus that encode a stress-activated protein kinase named “p54a” or“SAPKa” (Kyriakis et al., Nature, 1994, 369, 156). The structures ofthree control oligonucleotides, ISIS Nos. 21914 to 21916 (SEQ ID NOS:139to 141, respectively) are also shown in the table. Two isoforms of p54ahave been described: “p54a1” (GenBank accession No. L27112, locus name“RATSAPKA”) and “p54a2” (GenBank accession No. L27111, locus name“RATSAPKBD”). With the exception of ISIS 18257 (SEQ ID NO:128), theoligonucleotides described in Table 21 are specifically hybridizable tonucleic acids encoding either p54a1 or p54a2. ISIS 18257 is specificallyhybridizable to nucleic acids encoding p54a2 (i.e., GenBank accessionNo. L27112, locus name “RATSAPKB”). In Table 21, emboldened residues are2′-methoxyethoxy-residues (others are 2′-deoxy-); “C” residues are2′-methoxyethoxy-5-methyl-cytosines and “C” residues are5-methyl-cytosines; “o” indicates a phosphodiester linkage; and “s”indicates a phosphorothioate linkage. The target gene co-ordinates arefrom GenBank Accession No. L27112, locus name “RATSAPKB.” TABLE 21Nucleotide Sequences of Rat JNK2 Oligonucleotides SEQ TARGET GENE GENEISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ → 3′) NO:CO-ORDINATES REGION 18254 ToCoAoToGsAsTsGsTsAsGsTsGsTsCsAoToAoCoA 1250001-0020 tIR 18255 ToGoToGoGsTsGsTsGsAsAsCsAsCsAsToToToAoA 1260281-0300 ORF 18256 CoCoAoToAsTsGsAsAsTsAsAsCsCsTsGoAoCoAoT 1270361-0380 ORF 18257 GoAoToAoTsCsAsAsCsAsTsTsCsTsCsCoToToGoT 1280621-0640 ORF 18258 GoCoToToCsGsTsCsCsAsCsAsGsAsGsAoToCoCoG 1290941-0960 ORF 18259 GoCoToCoAsGsTsGsGsAsCsAsTsGsGsAoToGoAoG 1301201-1220 ORF 18260 AoToCoToGsCsGsAsGsGsTsTsTsCsAsToCoGoGoC 1311281-1300 tTR 18261 CoCoAoCoCsAsGsCsTsCsCsCsAsTsGsToGoCoToC 1321341-1360 3′-UTR 18262 CoAoGoToTsAsCsAsCsAsTsGsAsTsCsToGoToCoA 1331571-1590 3′-UTR 18263 AoAoGoAoGsGsAsTsTsAsAsGsAsGsAsToToAoToT 1341701-1720 3′-UTR 18264 AoGoCoAoGsAsGsTsGsAsAsAsTsAsCsAoAoCoToT 1352001-2020 3′-UTR 18265 ToGoToCoAsGsCsTsCsTsAsCsAsTsTsAoGoGoCoA 1362171-2190 3′-UTR 18266 AoGoToAoAsGsCsCsCsGsGsTsCsTsCsCoToAoAoG 1372371-2390 3′-UTR 18267 AoAoAoToGsGsAsAsAsAsGsGsAsCsAsGoCoAoGoC 1382405-2424 3′-UTR 21914 GoCoToCoAsGsTsGsGsAsTsAsTsGsGsAoToGoAoG 139 18259control — 21915 GoCoToAoAsGsCsGsGsTsCsAsAsGsGsToToGoAoG 140 18259control — 21916 GoCoToCoGsGsTsGsGsAsAsAsTsGsGsAoToCoAoG 141 18259control —

[0260] TABLE 22 Activities of Oligonucleotides Targeted to Rat JNK2 SEQGENE ISIS ID TARGET % % No: NO: REGION EXPRESSION INHIBITION control¹ —— 100% 0% 18254 125 tIR 20% 80% 18255 126 ORF 21% 79% 18256 127 ORF 80%20% 18257 128 ORF 32% 68% 18258 129 ORF 19% 81% 18259 130 ORF 15% 85%18260 131 ORF 41% 59% 18261 132 3′-UTR 47% 53% 18262 133 3′-UTR 50% 50%18263 134 3′-UTR 63% 37% 18264 135 3′-UTR 48% 52% 18265 136 3′-UTR 38%62% 18266 137 3′-UTR 66% 34% 18267 138 3′-UTR 84% 16%

[0261] These antisense compounds were tested for their ability tomodulate levels of p54a (JNK2) mRNA in A10 cells using the radiolabeledhuman JNK2 cDNA as a probe as described supra. The results are shown inTable 22. Oligonucleotides showing levels of inhibition from ≧ about 60%to about 100% of rat JNK2 mRNA levels include ISIS Nos. 18254, 18255,18257, 18258, 18259, 18260 and 18265 (SEQ ID NOS:125, 126, 128, 129,130, 131 and 136, respectively). These oligonucleotides are preferredembodiments of the invention for modulating rat JNK2 expression.Oligonucleotides showing levels of inhibition of rat JNK1 mRNAs by atleast 80% in this assay include ISIS Nos. 18254, 18255, 18258 and 18259(SEQ ID NOS:125, 126, 129 and 130, respectively). These oligonucleotidesare thus more preferred embodiments of the invention for modulating ratJNK2 expression. ISIS 18259 (SEQ ID NO:130) was chosen for use infurther studies (infra).

[0262] C. Dose Response

[0263] A dose response study was conducted using oligonucleotidestargeted to rat JNK1 (ISIS 21859; SEQ ID NO:113) and JNK2 (ISIS 18259;SEQ ID NO:130) and Northern assays. The results (Table 23) demonstratean increasing effect as the oligonucleotide concentration is raised andconfirm that ISIS Nos. 21859 and 18259 (SEQ ID NOS:113 and 130,respectively) specifically modulate levels of mRNA encoding JNK1 andJNK2, respectively. TABLE 23 Dose-Dependent Response to Rat JNKAntisense Oligonucleotides (ASOs) SEQ % % ISIS ID ASO EXPRESSIONEXPRESSION # NO: Description Dose JNK1 JNK2 active ASO  10 nM 74 101  50nM 25 98 100 nM 11 99 200 nM 8 101 18259 130 rat JNK2   0 nM 100 100active ASO  10 nM 95 81  50 nM 101 35 100 nM 94 15 200 nM 89 5

[0264] D. JNK3

[0265] Table 24 describes the sequences and structures of a set ofoligonucleotides, ISIS Nos. 21899 to 21912 (SEQ ID NOS:142 to 155,respectively) that were designed to be specifically hybridizable tonucleic acids from Rattus norvegicus that encode a stress-activatedprotein kinase named “p54β” that is homologous to the human protein JNK3(Kyriakis et al., Nature, 1994, 369, 156; GenBank accession No. L27128,locus name “RATSAPKC”). In Table 24, emboldened residues are2′-methoxyethoxy-residues (others are 2′-deoxy-); “C” residues are2′-methoxyethoxy-5-methyl-cytosines and “C” residues are5-methyl-cytosines; “o” indicates a phosphodiester linkage; and “s”indicates a phosphorothioate linkage. The target gene co-ordinates arefrom GenBank Accession No. L27128, locus name “RATSAPKC.” Theoligonucleotides are tested for their ability to modulate rat JNK3 mRNAlevels essentially according to the methods described in the precedingExamples.

[0266] In addition to being specifically hybridizable to nucleic acidsencoding rat JNK3, some of the oligonucleotides described in Table 24are also specifically hybridizable with JNK3-encoding nucleic acids fromhumans and Mus musculus (mouse). Table 25 sets out these relationships.These oligonucleotides are tested for their ability to modulate MRNAlevels of the human JNK genes according to the methods described inExample 5. TABLE 24 Nucleotide Sequences of Rat JNK3 OligonucleotidesSEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO.(5′ → 3′) NO: CO-ORDINATES REGION 21899GoGoGoCoTsTsTsCsAsTsTsAsGsCsCsAoCoAoToT 142 0021-0040 5′-UTR 21900GoGoToToGsGsTsTsCsAsCsTsGsCsAsGoToAoGoT 143 0241-0260 5′-UTR 21901ToGoCoToCsAsTsGsTsTsGsTsAsAsTsGoToToToG 144 0351-0370 tIR 21902GoToCoGoAsGsGsAsCsAsGsCsGsTsCsAoToAoCoG 145 0491-0510 ORF 21903CoGoAoCoAsTsCsCsGsCsTsCsGsTsGsGoToCoCoA 146 0731-0750 ORF 21904AoCoAoToAsCsGsGsAsGsTsCsAsTsCsAoToGoAoA 147 0901-0920 ORF 21905GoCoAoAoTsTsTsCsTsTsCsAsTsGsAsAoToToCoT 148 1101-1120 ORF 21906ToCoGoToAsCsCsAsAsAsCsGsTsTsGsAoToGoToA 149 1321-1340 ORF 21907CoGoCoCoGsAsGsGsCsTsTsCsCsAsGsGoCoToGoC 150 1601-1620 ORF 21908GoGoCoToAsGsTsCsAsCsCsTsGsCsAsAoCoAoAoC 151 1631-1650 tTR 21909GoCoGoToGsCsGsTsGsCsGsTsGsCsTsToGoCoGoT 152 1771-1790 3′-UTR 21910GoCoToCoAsGsCsTsGsCsGsAsTsAsCsAoGoAoAoC 153 1891-1910 3′-UTR 21911AoGoCoGoCsGsAsCsTsAsGsAsAsGsTsToAoAoGoT 154 1921-1940 3′-UTR 21912AoGoGoGoAsGsAsCsCsAsAsAsGsTsCsGoAoGoCoG 155 1941-1960 3′-UTR

[0267] TABLE 25 Cross-Hybridizations of Rat JNK3 O1igonuc1eotideHybridizes to: SEQ ISIS ID Human Human Mouse NO. NO: JNK3a1¹ JNK3a2²JNK3³ 21900 143 — —  bp 329-348 21901 144  bp 193-212  bp 169-188  bp411-430 21904 147 — —  bp 961-980 21905 148  bp 943-962  bp 919-938 —21906 149 — — bp 1381-1400 21908 151 bp 1478-1497 bp 1449-1468 bp1696-1715

[0268] E. Pan JNK Oligonucleotides

[0269] Certain of the oligonucleotides of the invention are capable ofmodulating two or more JNK proteins and are referred to herein as “PanJNK” oligonucleotides. For example, ISIS Nos. Nos. 21861 and 21867 (SEQID NOS:115 and 121, respectively) demonstrated a capacity to modulateboth JNK1 and JNK2 (Table 20). Such oligonucleotides are useful when theconcomitant modulation of several JNK proteins is desired.

[0270] Human Pan JNK oligonucleotides are described in Table 26. Theseoligonucleotides are designed to be complementary to sequences that areidentically conserved in (i.e., SEQ ID NOS:156, 158, 159, 160 and 161),or which occur with no more than a one-base mismatch (SEQ ID NO:157), innucleic acids encoding human JNK1a1, JNK1a2, JNK2a1 and JNK2a2. Theoligonucleotides described in Table 26 are evaluated for their abilityto modulate JNK1 and JNK2 mRNA levels in A549 cells using the methodsand assays described in Examples 3 and 4.

[0271] In instances where such common sequences encompass one or morebase differences between the JNK genes that it is desired to modulate,hypoxanthine (inosine) may be incorporated at the positions of theoligonucleotide corresponding to such base differences. (“Hypoxanthine”is the art-accepted term for the base that corresponds to the nucleosideinosine; however, the term “inosine” is used herein in accordance withU.S. and PCT rules regarding nucleotide sequences.) As is known in theart, inosine (I) is capable of hydrogen bonding with a variety ofnucleobases and thus serves as a “universal” base for hybridizationpurposes. For example, an oligonucleotide having a sequence that is aderivative of SEQ ID NO:157 having one inosine substitution(TAGGAIATTCTTTCATGATC, SEQ ID NO:162) is predicted to bind to nucleicacids encoding human JNK1a1, JNK1a2, JNK2a1 and JNK2a2 with nomismatched bases. As another example, an oligonucleotide having asequence that is a derivative of SEQ ID NO:161 having one inosinesubstitution (GGTTGCAITTTCTTCATGAA, SEQ ID NO:163) is predicted to bindwith no mismatched bases to nucleic acids encoding human JNK3al andJNK3a2 in addition to JNK1a1, JNK1a2, JNK2a1 and JNK2a2. Sucholigonucleotides are evaluated for their ability to modulate JNK1 andJNK2 mRNA levels in A549 cells, and JNK3 mRNA levels in SH-SY5Y cells,using the methods and assays described in Examples 3, 4 and 5. TABLE 26Human Pan JNK Oligonucleotides NUCLEOTIDE SEQUENCE (5′ → 3′) ANDCHEMICAL MODIFICATIONS* SEQ ID NO: A ^(S) C ^(S) A ^(S) T ^(S) C ^(S) T^(S)T^(O)G^(O)A^(O)A^(O)A^(O)T^(O)T^(O)C^(S) T ^(S) T ^(S) C ^(S) T ^(S)A ^(S) G 156 T ^(S) A ^(S) G ^(S) G ^(S) A ^(S) T^(S)A^(O)T^(O)T^(O)C^(O)T^(O)T^(O)T^(O)C^(S) A ^(S) T ^(S) G ^(S) A ^(S)T ^(S) C 157 A ^(S) G ^(S) A ^(S) A ^(S) G ^(S) G^(S)T^(O)A^(O)G^(O)G^(O)A^(O)C^(O)A^(O)T^(S) T ^(S) C ^(S) T ^(S) T ^(S)T ^(S) C 158 T ^(S) T ^(S) T ^(S) A ^(S) T ^(S) T^(S)C^(O)C^(O)A^(O)C^(O)T^(O)G^(O)A^(O)T^(S) C ^(S) A ^(S) A ^(S) T ^(S)A ^(S) T 159 T ^(S) C ^(S) A ^(S) A ^(S) T ^(S) A^(S)A^(O)C^(O)T^(O)T^(O)T^(O)A^(O)T^(O)T^(S) C ^(S) C ^(S) A ^(S) C ^(S)T ^(S) G 160 G ^(S) G ^(S) T ^(S) T ^(S) G ^(S) C^(S)A^(O)G^(O)T^(O)T^(O)T^(O)C^(O)T^(O)T^(S) C ^(S) A ^(S) T ^(S) G ^(S)A ^(S) A 161

Example 9 Effect of Oligonucleotides Targeted to Human JNK1 and JNK2 onTNFa-induced JNK Activity

[0272] Human umbilical vein endothelial cells (HUVEC, Clonetics, SanDiego Calif.) were incubated with oligonucleotide with LipofectinJ inOpti-MEMJ for 4 hours at 371 C./5% Co₂. The medium was then replacedwith 1% FBS/EGM (Clonetics, Walkersville Md.) and incubated for 24 hoursat 371 C./5% CO₂. Cells were treated with 5 ng/ml TNFa for 15 minutesbefore lysis. JNK activity was determined by incubating lysates(normalized for protein) with immobilized GST-c-Jun fusion protein(e.g., New England Biolabs, Beverly, Mass.)+?³²P-ATP. GST-c-Jun beadswere washed and SDS-PAGE sample buffer was added. Samples were resolvedby SDS-PAGE and phosphorylated c-Jun was visualized using a MolecularDynamics PhosphorImager.

[0273] Compared to a control oligonucleotide, the JNK1 oligonucleotideISIS 15346 (SEQ ID NO: 16; 100 nM concentration)inhibited TNFa-inducedJNK activity by approximately 70%. The JNK2 oligonucleotide ISIS 15353(SEQ ID NO: 31; 100 nM) inhibited TNFa-induced JNK activity byapproximately 55%. A combination of 50 nM each oligonucleotide inhibitedTNFa-induced JNK activity by approximately 68% and a combination of 100nM each oligonucleotide inhibited TNFa-induced JNK activity byapproximately 83%.

Example 10 Effect of Oligonucleotides Targeted to Human JNK1 and JNK2 onApoptosis

[0274] TNFa causes apoptosis in many cell types. The effect of JNK1 orJNK2 antisense oligonucleotides on TNFa-induced apoptosis in HUVEC wasexamined. HUVEC were incubated with oligonucleotides in Opti-MEMJ plusLipofectinJ for four hours at 371 C./5% CO₂. The medium was thenreplaced with 1% FBS/EGM (Clonetics, Walkersville Md.) and incubated for44 hours at 371 C./5% CO₂. Cells were treated with 10 ng/ml TNFa with orwithout 10 μg/ml cyclohexamide or 100 mM z-VAD.fmk (a caspase inhibitor;Calbiochem, La Jolla Calif.) and incubated for 24 hours at 371 C./5%CO₂. Cells were collected using trypsin/EDTA, washed and fixed in 70%ethanol. Cells were stained with propidium iodide and analyzed for DNAcontent by flow cytometry. Results are shown in Table 27, expressed aspercent hypodiploid cells, a measure of apoptosis. Controloligonucleotides are: ISIS 18076 (CTTTCCGTTGGACCCCTGGG; SEQ ID NO:164),scrambled control for ISIS 15346. ISIS 18078 (GTGCGCGCGAGCCCGAAATC; SEQID NO:165), scrambled control for ISIS 15353. Both are 2′-methoxyethoxygapmers with phosphorothioate backbone linkages throughout. TABLE 27Effect of antisense inhibitors of JNK1 and JNK2 on apoptosis Numbersgiven are percent hypodiploid cells (a measure of apoptosis) JNK1 JNK1JNK2 JNK2 AS control AS control No (ISIS (ISIS (ISIS (ISIS oligo 15346)18076) 15353) 18078) Oligo alone 14 13 8 23 11 Oligo + TNFa 15 14 10 3211 Oligo + 7 8 11 27 12 Cyclohexamide Oligo + 5 5 10 17 9 z-VAD.fmk

[0275] It can be seen from the table that antisense suppression of JNK1or JNK2 expression had little effect on resistance to TNFa-inducedapoptosis. However, it was found that antisense inhibition of JNK2, butnot JNK1, resulted in increased cell death even in the absence of TNFa,suggesting that JNK2 may play a role in protecting these cells fromapoptosis. JNK2 oligonucleotide-induced cell death was decreased by thecaspase inhibitor z-FAD.fmk, suggesting that caspase activation wasinvolved in this apoptotic response. Protein synthesis was not believedto be required because cyclohexamide, an inhibitor of protein synthesis,had no effect on apoptosis after JNK2 oligonucleotide treatment.

Example 11 Effect of Oligonucleotides Targeted to Human JNK2 on ProstateCancer

[0276] Human JNK2 antisense oligonucleotides were used in a human tumorxenograft model to determine the effectiveness of treating prostatecancer. In advanced prostate cancer, progression to androgenindependence occurs. JNK activation of AP-1 can modulate expression ofthe androgen receptor.

[0277] LNCaP cells (human prostate cancer cells purchased from AmericanType Culture Collection, Rockville, Md.) were maintained in RPMI 1640(Terry Fox Laboratory, Vancouver, BC, Canada) with 5% fetal bovine serum(GIBCO, Burlington, ON, Canada). Six to eight week old male athymic nudemice (BALB/c strain) were purchased from Harlan Sprague Dawley, Inc.(Indianapolis, Ind.).

[0278] 1×10⁶ LNCaP cells were inoculated subcutaneously with 0.1 mlMATRIGEL (Becton Dickinson Labware, Bedford, Mass.) in the flank regionof the mice. Blood samples were obtained with tail vein incisions ofmice and serum PSA levels were determined by an enzymatic immunoassaykit (Abbott IMX, Montreal, PQ, Canada) according to the manufacturer'sprotocol. When serum PSA levels increased to around 100 ng/ml, 4-6 weekspost-injection, mice were castrated via a scrotal approach. Mice weretreated with 12.5 mg/kg oligonucleotide intraperitoneally once daily,beginning one day after castration. Tumor volume and serum PSA levelswere measured once weekly.

[0279] Results are shown in Table 28. LNCaP tumor growth rates were 2.5fold higher in the control group compared to JNK2 antisense group. Tumorvolume in the control group increased twofold above baseline by 7 weekspost-castration, and 5-fold by 10 weeks post-castration. In contrast,mean tumor volume in the JNK2 antisense-treated group decreased aftercastration to 60% of baseline by 4 weeks post-castration and returned topre-castration level by 7 weeks post-castration. Thereafter, mean tumorvolume increased to twofold above baseline by 10 weeks post-castrationcompared to 5-fold in the control oligonucleotide-treated group.

[0280] Differences in serum PSA between the JNK2 antisense-treated miceand control oligonucleotide-treated mice were clear. By one weekpost-castration, serum PSA decreased by 67% and 89% in the controloligonucleotide and JNK2 antisense oligonucleotide-treated groups,respectively to nadir levels by one week post-castration. In the controloligonucleotide treated group, mean serum PSA increased beginning twoweeks post-castration and returned to pre-castration level by 5 weekspost-castration, a response typical of castrated control mice. Sato etal., J. Steroid Biochem. Molec. Biol., 1996, 58, 139-146. By ten weekspost-castration, mean serum PSA increased to threefold above baselinelevels. In contrast, in JNK2 antisense treated mice, mean serum PSAremained at or below baseline levels at 10 weeks post-castration.

[0281] Time to progression to androgen-independent PSA regulation wasdefined as the duration of time after castration for serum PSA levels toreturn to levels equal to or greater than pre-castration levels. Datapoints were expressed as average PSA levels ± standard error of the meanbased on seven measurements. The time to progression to androgenindependence after castration was delayed in the mice treated with JNK2antisense by 100% (10 weeks vs. 5 weeks for control group). Nosignificant toxicity was observed in any treatment group during the 10week treatment period. TABLE 28 JNK2 Antisense Oligonucleotides inProstate Cancer SEQ Weeks Tumor Serum PSA ID Gene Post- volume levelsISIS # NO: Target castration (mm³) (ng/ml) 9 15353 31 JNK2 0 203 84 ″ ″″ 1 149 9 ″ ″ ″ 2 119 11 ″ ″ ″ 3 128 10 ″ ″ ″ 4 118 15 ″ ″ ″ 5 159 20 ″″ ″ 5 159 20 ″ ″ ″ 6 166 25 ″ ″ ″ 7 196 34 ″ ″ ″ 8 239 47 ″ ″ ″ 9 307 63″ ″ ″ 10 422 87 14616 control 0 228 82 ″ ″ ″ 1 177 27 ″ ″ ″ 2 235 33 ″ ″″ 3 207 51 ″ ″ ″ 4 261 77 ″ ″ ″ 5 274 85 ″ ″ ″ 6 316 113 ″ ″ ″ 7 453 129″ ″ ″ 8 514 154 ″ ″ ″ 9 699 210 ″ ″ ″ 10 1020 257

Example 12 Inhibition of Inflammatory Responses by AntisenseOligonucleotides Targeting JNK Family Members

[0282] JNKs have been implicated as key mediators of a variety ofcellular responses and pathologies. JNKs can be activated byenvironmental stress, such as radiation, heat shock, osmotic shock, orgrowth factor withdrawal as well as by pro-inflammatory cytokines.

[0283] Antisense oligonucleotides targeting any of the JNK familymembers described in Examples 3-5 are synthesized and purified as inExample 1 and evaluated for their activity in inhibiting inflammatoryresponses. Such inhibition is evident in the reduction of production ofpro-inflammatory molecules by inflammatory cells or upon the attenuationof proliferation of infiltrating or inflammatory cells, the mostprominent of which are lymphocytes, neutrophils, macrophages andmonocytes. Following synthesis, oligonucleotides are tested in anappropriate model system using optimal tissue or cell cultureconditions. Inflammatory cells including lymphocytes, neutrophils,monocytes and macrophages are treated with the antisenseoligonucleotides by the method of electroporation. Briefly, cells (5×10⁶cells in PBS) are transfected with oligonucleotides by electroporationat 200V, 1000 uF using a BTX Electro Cell Manipulator 600 (Genetronics,San Diego, Calif.). For an initial screen, cells are electroporated with10 uM oligonucleotide and RNA is collected 24 hours later. Controlswithout oligonucleotide are subjected to the same electroporationconditions.

[0284] Total cellular RNA is then isolated using the RNEASY7 kit(Qiagen, Santa Clarita, Calif.). RNAse protection experiments areconducted using RIBOQUANT™ kits and template sets according to themanufacturer's instructions (Pharmingen, San Diego, Calif.).

[0285] Adherent cells such as endothelial and A549 cells are transfectedusing the LIPOFECTIN™ protocol described in Example 2. Reduced JNK mRNAexpression is measured by Northern analysis while protein expression ismeasured by Western blot analysis, both described in Example 1. Negativecontrol oligonucleotides with mismatch sequences are used to establishbaselines and non-specific effects.

[0286] The degree of inflammatory response is measured by determiningthe levels of inflammatory cytokine expression by Northern or Westernanalysis, or cytokine secretion by enzyme-linked immunosorbent assay(ELISA) techniques. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

[0287] The degree of inflammatory response is also determined bymeasuring the expression of known immediate-early genes by the method ofNorthern or Western blot analysis. Further into the inflammatoryresponse, levels of apoptosis are measured by flow cytometry asdescribed in Example 10.

Example 13 Inhibition of Fibrosis by Antisense OligonucleotidesTargeting JNK Family Members

[0288] Pulmonary fibrosis is characterized by inflammatory andfibroproliferative changes in the lung and an excess accumulation ofcollagen in the interstitium. There is also an increased recruitment ofimmune and inflammatory cells to the lung which act not only in theinitial damage to the lung but in the progression of the fibroticprocess.

[0289] In the rodent bleomycin (BL)-induced pulmonary fibrosis model,inhibition of fibrosis in the lung is determined by measuring any ofseveral markers for the condition. The BL-induced model is widelyaccepted in the art and can be found at, for example, Thrall, R. S. etal., Bleomycin In: Pulmonary Fibrosis, pp. 777-836, Eds. Phan, S. H. andThrall, R. S., Marcel Dekker, New York, 1995 and Giri, S. N. et al.,Miscellaneous mediator systems in pulmonary fibrosis In: PulmonaryFibrosis, pp. 231-292, Eds. Phan, S. H. and Thrall, R. S., MarcelDekker, New York, 1995.

[0290] Antisense oligonucleotides targeting any of the JNK familymembers described in Examples 3-5 are synthesized and purified as inExample 1 and evaluated for their ability to prevent or inhibitpulmonary fibrosis. These fibrotic markers include release of variouspro-inflammatory mediators including cytokines and chemokines such asTNFa, interleukin-8 and interleukin-6, increased numbers of proteasesand metalloproteinases, generation of reactive oxygen species (ROS),edema, hemorrhage and cellular infiltration predominated by neutrophilsand macrophages.

[0291] Following synthesis, oligonucleotides are tested in the rodentBL-induced pulmonary fibrosis model using optimal conditions. Micereceive an intratracheal dose of bleomycin (0.125 U/mouse) or saline,followed by treatment with antisense oligonucleotide (i.p.) over 2weeks. After 2 weeks mice are sacrificed and biochemical,histopathological and immunohistochemical analyses are performed.

[0292] Biochemical and immunohistochemical analysis involves themeasurement of the levels of pro-inflammatory cytokine expression byNorthern or Western analysis, or cytokine secretion by enzyme-linkedimmunosorbent assay (ELISA) techniques as described in Example 12.Histopathological analyses are performed for the presense of fibroticlesions in the BL-treated lungs and for the presence of and number ofcells with the fibrotic phenotype by methods which are standard in theart.

Example 14 Sensitization to Chemotherapeutic Agents by AntisenseOligonucleotides Targeting JNK Family Members

[0293] Manipulation of cancer chemotherapeutic drug resistance can alsobe accomplished using antisense oligonucleotides targeting JNK familymembers. Antisense oligonucleotides targeting any of the JNK familymembers described in Examples 3-5 are synthesized and purified as inExample 1 and evaluated for their ability to sensitize cells to theeffects of chemotherapeutic agents. Sensitization is evident in theincreased number of target cells undergoing apoptosis subsequent totreatment.

[0294] Following synthesis, oligonucleotides are tested in anappropriate model system using optimal tissue or cell cultureconditions. Cells are treated with the compounds of the invention inconjunction with one or more chemotherapeutic agents in a treatmentregimen wherein the chemotherapeutic agents may be used individually(e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU andoligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide).

[0295] For nonadherent cells, treatment is by the method ofelectroporation. Briefly, cells (5×10⁶ cells in PBS) are transfectedwith oligonucleotides by electroporation either before, during or aftertreatment with the chemotherapeutic agent, at 200V, 1000 uF using a BTXElectro Cell Manipulator 600 (Genetronics, San Diego, Calif.). For aninitial screen, cells are electroporated with 10 uM oligonucleotide andRNA is collected 24 hours later. Controls without oligonucleotide orchemotherapeutic agent are subjected to the same electroporationconditions.

[0296] Total cellular RNA is then isolated using the RNEASY7 kit(Qiagen, Santa Clarita, Calif.). RNAse protection experiments areconducted using RIBOQUAN™ kits and template sets according to themanufacturer's instructions (Pharmingen, San Diego, Calif.).

[0297] Adherent cells such as endothelial and A549 cells are transfectedusing the LIPOFECTIN™ protocol described in Example 2. Reduced JNK mRNAexpression is measured by Northern analysis while protein expression ismeasured by Western blot analysis, both described in Example 1. Negativecontrol oligonucleotides with mismatch sequences can be used toestablish baselines and non-specific effects.

[0298] The degree of apoptosis, and consequently sensitization ismeasured by flow cytometry as described in Example 10.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 165 <210> SEQ ID NO 1<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 1 attctttcca ctcttctatt 20 <210> SEQ ID NO 2 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 2 ctcctccaagtccataactt 20 <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 3 cccgtataac tccattcttg 20 <210> SEQID NO 4 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence<400> SEQUENCE: 4 ctgtgctaaa ggagagggct 20 <210> SEQ ID NO 5 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 5atgatggatg ctgagagcca 20 <210> SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 6 gttgacattg aagacacatc20 <210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 7 ctgtatcaga ggccaaagtc 20 <210> SEQ ID NO 8<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 8 tgctgcttct agactgctgt 20 <210> SEQ ID NO 9 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 9 agtcatctacagcagcccag 20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 10 ccatccctcc caccccccga 20 <210> SEQID NO 11 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence<400> SEQUENCE: 11 atcaatgact aaccgactcc 20 <210> SEQ ID NO 12 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 12caaaaataag accactgaat 20 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 13 cacgcttgct tctgctcatg20 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 14 cggcttagct tcttgattgc 20 <210> SEQ ID NO 15<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 15 cccgcttggc atgagtctga 20 <210> SEQ ID NO 16 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 16ctctctgtag gcccgcttgg 20 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 17 atttgcatcc atgagctcca20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 18 cgttcctgca gtcctggcca 20 <210> SEQ ID NO 19<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 19 ggatgacctc gggtgctctg 20 <210> SEQ ID NO 20 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 20cccataatgc accccacaga 20 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 21 cgggtgttgg agagcttcat20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 22 tttggtggtg gagcttctgc 20 <210> SEQ ID NO 23<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 23 ggctgccccc gtataactcc 20 <210> SEQ ID NO 24 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 24tgctaaagga gagggctgcc 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 25 aggccaaagt cggatctgtt20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 26 ccaccccccg atggcccaag 20 <210> SEQ ID NO 27<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 27 ccaagcgggc ctacagagag 20 <210> SEQ ID NO 28 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 28ctttccgttg gacccctggg 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 29 gtttcagatc cctcgcccgc20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 30 tgcagcacaa acaatccctt 20 <210> SEQ ID NO 31<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 31 gtccgggcca ggccaaagtc 20 <210> SEQ ID NO 32 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 32caggatgact tcgggcgccc 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 33 gctctcccat gatgcaaccc20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 34 atgggtgacg cagagcttcg 20 <210> SEQ ID NO 35<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 35 ctgctgcatc tgaaggctga 20 <210> SEQ ID NO 36 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 36tgagaaggag tggcgttgct 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 37 tgctgtctgt gtctgaggcc20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 38 ggtcccgtcg aggcatcaag 20 <210> SEQ ID NO 39<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 39 catttcaggc ccacggaggt 20 <210> SEQ ID NO 40 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 40ggtctgaata gggcaaggca 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 41 gggcaagtcc aagcaagcat20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 42 gactttggcc tggcccggac 20 <210> SEQ ID NO 43<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 43 gtgcgcgcga gcccgaaatc 20 <210> SEQ ID NO 44 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 44ttcaacagtt tcttgcataa 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 45 ctcatctata ggaaacgggt20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 46 tggaggctca taaataccac 20 <210> SEQ ID NO 47<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 47 tataagaaat ggaggctcat 20 <210> SEQ ID NO 48 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 48tcacatccaa tgttggttca 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 49 ttatcgaatc cctgacaaaa20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 50 gtttggcaat atatgacaca 20 <210> SEQ ID NO 51<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 51 ctgtcaagga cagcatcata 20 <210> SEQ ID NO 52 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 52aatcacttga cataagttgg 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 53 taaatccctg tgaataattc20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 54 gcatcccaca gaccatatat 20 <210> SEQ ID NO 55<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 55 tgttctcttt catccaactg 20 <210> SEQ ID NO 56 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 56tctcactgct gttcactgct 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 57 gggtctggtc ggtggacatg20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 58 aggctgctgt cagtgtcaga 20 <210> SEQ ID NO 59<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 59 tcacctgcaa caacccaggg 20 <210> SEQ ID NO 60 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 60gcggctagtc acctgcaaca 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 61 cgctgggttt cgcaggcagg20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 62 atcatctcct gaagaacgct 20 <210> SEQ ID NO 63<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Homo Sapiens <300>PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: L26318 Genbank<309> DATABASE ENTRY DATE: 1994-04-25 <313> RELEVANT RESIDUES: FROM 631TO 665 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:U34822 Genbank <309> DATABASE ENTRY DATE: 1996-07-26 <313> RELEVANTRESIDUES: FROM 625 TO 659 <400> SEQUENCE: 63 aacgtggatt tatggtctgtggggtgcatt atggg 35 <210> SEQ ID NO 64 <211> LENGTH: 35 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <308>DATABASE ACCESSION NUMBER: U35004 Genbank <309> DATABASE ENTRY DATE:1996-07-26 <313> RELEVANT RESIDUES: FROM 631 TO 665 <300> PUBLICATIONINFORMATION: <308> DATABASE ACCESSION NUMBER: U35005 Genbank <309>DATABASE ENTRY DATE: 1996-07-26 <313> RELEVANT RESIDUES: FROM 626 TO 660<400> SEQUENCE: 64 aacgttgaca tttggtcagt tgggtgcatc atggg 35 <210> SEQID NO 65 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence<400> SEQUENCE: 65 cccataatgc accccacaga ccataaatcc acgtt 35 <210> SEQID NO 66 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence<400> SEQUENCE: 66 cccatgatgc acccaactga ccaaatgtca acgtt 35 <210> SEQID NO 67 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: L26318Genbank <309> DATABASE ENTRY DATE: 1994-04-25 <313> RELEVANT RESIDUES:FROM 668 TO 711 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: U34822 Genbank <309> DATABASE ENTRY DATE: 1996-07-26 <313>RELEVANT RESIDUES: FROM 662 TO 705 <400> SEQUENCE: 67 aaatggtttgccacaaaatc ctctttccag gaagggacta tatt 44 <210> SEQ ID NO 68 <211>LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300>PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: U35004 Genbank<309> DATABASE ENTRY DATE: 1996-07-26 <313> RELEVANT RESIDUES: FROM 668TO 711 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:U35005 Genbank <309> DATABASE ENTRY DATE: 1996-07-26 <313> RELEVANTRESIDUES: FROM 663 TO 706 <400> SEQUENCE: 68 aaatgatcaa aggtggtgttttgttcccag gtacagatca tatt 44 <210> SEQ ID NO 69 <211> LENGTH: 44 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 69 aatatagtcc cttcctggaaagaggatttt gtggcaaacc attt 44 <210> SEQ ID NO 70 <211> LENGTH: 44 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 70 aatatgatct gtacctgggaacaaaacacc acctttgatc attt 44 <210> SEQ ID NO 71 <211> LENGTH: 32 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <308> DATABASE ACCESSION NUMBER:L26318 Genbank <309> DATABASE ENTRY DATE: 1994-04-25 <313> RELEVANTRESIDUES: FROM 1144 TO 1175 <300> PUBLICATION INFORMATION: <308>DATABASE ACCESSION NUMBER: U35004 Genbank <309> DATABASE ENTRY DATE:1996-07-26 <313> RELEVANT RESIDUES: FROM 1144 TO 1175 <400> SEQUENCE: 71ccctctcctt tagcacaggt gcagcagtga tc 32 <210> SEQ ID NO 72 <211> LENGTH:27 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300> PUBLICATIONINFORMATION: <308> DATABASE ACCESSION NUMBER: U34822 Genbank <309>DATABASE ENTRY DATE: 1996-07-26 <313> RELEVANT RESIDUES: FROM 1138 TO1164 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:U35005 Genbank <309> DATABASE ENTRY DATE: 1996-07-26 <313> RELEVANTRESIDUES: FROM 1139 TO 1165 <400> SEQUENCE: 72 ccctctcctt taggtgcagcagtgatc 27 <210> SEQ ID NO 73 <211> LENGTH: 32 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 73 gatcactgct gcacctgtgc taaaggagaggg 32 <210> SEQ ID NO 74 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 74 gatcactgct gcacctaaag gagaggg 27<210> SEQ ID NO 75 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: Homosapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:L31951 Genbank <309> DATABASE ENTRY DATE: 1994-12-06 <313> RELEVANTRESIDUES: FROM 689 TO 748 <300> PUBLICATION INFORMATION: <308> DATABASEACCESSION NUMBER: U34821 Genbank <309> DATABASE ENTRY DATE: 1996-07- 26<313> RELEVANT RESIDUES: FROM 675 TO 734 <400> SEQUENCE: 75 gtgggttgcatcatgggaga gctggtgaaa ggttgtgtga tattccaagg 50 cactgaccat 60 <210> SEQID NO 76 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: U35002Genbank <309> DATABASE ENTRY DATE: 1994-07-26 <313> RELEVANT RESIDUES:FROM 653 TO 712 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: U35003 Genbank <309> DATABASE ENTRY DATE: 1996-07- 26 <313>RELEVANT RESIDUES: FROM 665 TO 724 <400> SEQUENCE: 76 gtcgggtgcatcatggcaga aatggtcctc cataaagtcc tgttcccggg 50 aagagactat 60 <210> SEQID NO 77 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence<400> SEQUENCE: 77 atggtcagtg ccttggaata tcacacaacc tttcaccagctctcccatga 50 tgcaacccac 60 <210> SEQ ID NO 78 <211> LENGTH: 60 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 78 atagtctctt cccgggaacaggactttatg gaggaccatt tctgccatga 50 tgcacccgac 60 <210> SEQ ID NO 79<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300>PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: U34821 Genbank<309> DATABASE ENTRY DATE: 1996-07- 26 <313> RELEVANT RESIDUES: FROM1164 TO 1198 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: U35002 Genbank <309> DATABASE ENTRY DATE: 1994-07-26 <313>RELEVANT RESIDUES: FROM 1142 TO 1176 <400> SEQUENCE: 79 gatcagccttcagcacagat gcagcagtaa gtagc 35 <210> SEQ ID NO 80 <211> LENGTH: 30 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION:<308> DATABASE ACCESSION NUMBER: L31951 Genbank <309> DATABASE ENTRYDATE: 1994-12-06 <313> RELEVANT RESIDUES: FROM 1178 TO 1207 <300>PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: U35003 Genbank<309> DATABASE ENTRY DATE: 1996-07- 26 <313> RELEVANT RESIDUES: FROM1154 TO 1183 <400> SEQUENCE: 80 gatcagcctt cagatgcagc agtaagtagc 30<210> SEQ ID NO 81 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 81 gctacttact gctgcatctg tgctgaaggc tgatc 35<210> SEQ ID NO 82 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 82 gctacttact gctgcatctg aaggctgatc 30 <210>SEQ ID NO 83 <211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Homosapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:U34820 Genbank <309> DATABASE ENTRY DATE: 1994-07-26 <313> RELEVANTRESIDUES: FROM 1325 TO 1362 <400> SEQUENCE: 83 ggacagcctt ctccttcagcacaggtgcag cagtgaac 38 <210> SEQ ID NO 84 <211> LENGTH: 33 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <308>DATABASE ACCESSION NUMBER: U34819 Genbank <309> DATABASE ENTRY DATE:1994-07-26 <313> RELEVANT RESIDUES: FROM 1301 TO 1333 <400> SEQUENCE: 84ggacagcctt ctccttcagg tgcagcagtg aac 33 <210> SEQ ID NO 85 <211> LENGTH:38 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 85gttcactgct gcacctgtgc tgaaggagaa ggctgtcc 38 <210> SEQ ID NO 86 <211>LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 86gttcactgct gcacctgaag gagaaggctg tcc 33 <210> SEQ ID NO 87 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 87atgggtgact cagagcttcg 20 <210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 88 atgggttact cagagcttcg20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 89 atgggttact catagcttcg 20 <210> SEQ ID NO 90<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 90 atgtgttact catagcttcg 20 <210> SEQ ID NO 91 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 91ttgtgttact catagcttcg 20 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 92 ttgtgttact catagtttcg20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 93 ctgctgcatt tgaaggctga 20 <210> SEQ ID NO 94<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 94 ctgctgcatt tgtaggctga 20 <210> SEQ ID NO 95 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 95ctgctgtatt tgtaggctga 20 <210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 96 ctgttgtatt tgtaggctga20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 97 ctgttgtatt tgtagtctga 20 <210> SEQ ID NO 98<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 98 ttgttgtatt tgtagtctga 20 <210> SEQ ID NO 99 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 99tgctgtctga gtctgaggcc 20 <210> SEQ ID NO 100 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 100 tgctgtatgagtctgaggcc 20 <210> SEQ ID NO 101 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 101 tgctgtatga gtatgaggcc 20 <210>SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 102 tgcagtatga gtatgaggcc 20 <210> SEQ ID NO103 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 103 tgcagtatga gtatgaagcc 20 <210> SEQ ID NO 104 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 104agcagtatga gtatgaagcc 20 <210> SEQ ID NO 105 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 105 ggtcccgtctaggcatcaag 20 <210> SEQ ID NO 106 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 106 ggtcccttct aggcatcaag 20 <210>SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 107 ggttccttct aggcatcaag 20 <210> SEQ ID NO108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 108 ggttccttct agtcatcaag 20 <210> SEQ ID NO 109 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 109ggttccttct agtcattaag 20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 110 tgttccttctagtcattaag 20 <210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 111 caacgtcccg cgctcggccg 20 <210>SEQ ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 112 cctgctcgc ggctccgcgtt 20 <210> SEQ ID NO113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 113 ctcatgatgg caagcaatta 20 <210> SEQ ID NO 114 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 114tgttgtcacg tttacttctg 20 <210> SEQ ID NO 115 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 115 cggtaggctcgcttagcatg 20 <210> SEQ ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 116 ctagggattt ctgtggtgtg 20 <210>SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 117 cagcagagtg aaggtgcttg 20 <210> SEQ ID NO118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: : <400> SEQUENCE: 118 tcgttcctgcagtccttgcc 20 <210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 119 ccatttctcc cataatgcac 20 <210>SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 120 tgaattcagg acaaggtgtt 20 <210> SEQ ID NO121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 121 agcttcgtct acggagatcc 20 <210> SEQ ID NO 122 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 122cactcctcta ttgtgtgctc 20 <210> SEQ ID NO 123 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 123 gctgcacctaaaggagacgg 20 <210> SEQ ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 124 ccagagtcgg atctgtggac 20 <210>SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 125 tcatgatgta gtgtcataca 20 <210> SEQ ID NO126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 126 tgtggtgtga acacatttaa 20 <210> SEQ ID NO 127 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 127ccatatgaat aacctgacat 20 <210> SEQ ID NO 128 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 128 gatatcaacattctccttgt 20 <210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 129 gcttcgtcca cagagatccg 20 <210>SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 130 gctcagtgga catggatgag 20 <210> SEQ ID NO131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 131 atctgcgagg tttcatcggc 20 <210> SEQ ID NO 132 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 132ccaccagctc ccatgtgctc 20 <210> SEQ ID NO 133 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 133 cagttacacatgatctgtca 20 <210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 134 aagaggatta agagattatt 20 <210>SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 135 agcagagtga aatacaactt 20 <210> SEQ ID NO136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 136 tgtcagctct acattaggca 20 <210> SEQ ID NO 137 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 137agtaagcccg gtctcctaag 20 <210> SEQ ID NO 138 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 138 aaatggaaaaggacagcagc 20 <210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 139 gctcagtgga tatggatgag 20 <210>SEQ ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 140 gctaagcggt caaggttgag 20 <210> SEQ ID NO141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 141 gctcggtgga aatggatcag 20 <210> SEQ ID NO 142 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 142gggctttcat tagccacatt 20 <210> SEQ ID NO 143 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 143 ggttggttcactgcagtagt 20 <210> SEQ ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 144 tgctcatgtt gtaatgtttg 20 <210>SEQ ID NO 145 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 145 gtcgaggaca gcgtcatacg 20 <210> SEQ ID NO146 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 146 cgacatccgc tcgtggtcca 20 <210> SEQ ID NO 147 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 147acatacggag tcatcatgaa 20 <210> SEQ ID NO 148 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 148 gcaatttcttcatgaattct 20 <210> SEQ ID NO 149 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 149 tcgtaccaaa cgttgatgta 20 <210>SEQ ID NO 150 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 150 cgccgaggct tccaggctgc 20 <210> SEQ ID NO151 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 151 ggctagtcac ctgcaacaac 20 <210> SEQ ID NO 152 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 152gcgtgcgtgc gtgcttgcgt 20 <210> SEQ ID NO 153 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 153 gctcagctgcgatacagaac 20 <210> SEQ ID NO 154 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 154 agcgcgacta gaagttaagt 20 <210>SEQ ID NO 155 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 155 agggagacca aagtcgagcg 20 <210> SEQ ID NO156 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 156 acatcttgaa attcttctag 20 <210> SEQ ID NO 157 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 157taggatattc tttcatgatc 20 <210> SEQ ID NO 158 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 158 agaaggtaggacattctttc 20 <210> SEQ ID NO 159 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic Sequence <400> SEQUENCE: 159 tttattccac tgatcaatat 20 <210>SEQ ID NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticSequence <400> SEQUENCE: 160 tcaataactt tattccactg 20 <210> SEQ ID NO161 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Sequence <400>SEQUENCE: 161 ggttgcagtt tcttcatgaa 20 <210> SEQ ID NO 162 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <221> NAME/KEY:modified_base <222> LOCATION: 6 <223> OTHER INFORMATION: n=inosine <400>SEQUENCE: 162 tagganattc tttcatgatc 20 <210> SEQ ID NO 163 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <221> NAME/KEY:modified_base <222> LOCATION: 8 <223> OTHER INFORMATION: n=inosine <400>SEQUENCE: 163 ggttgcantt tcttcatgaa 20 <210> SEQ ID NO 164 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Sequence <400> SEQUENCE: 164ctttccgttg gacccctggg 20 <210> SEQ ID NO 165 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic Sequence <400> SEQUENCE: 165 gtgcgcgcgagcccgaaatc 20

What is claimed is:
 1. An oligonucleotide comprising from 8 to 30nucleotides connected by covalent linkages, wherein said oligonucleotidehas a sequence specifically hybridizable with a nucleic acid encoding aJNK protein and said oligonucleotide modulates the expression of saidJNK protein.
 2. The oligonucleotide of claim 1, wherein at least one ofsaid covalent linkages of said oligonucleotide is a modified covalentlinkage.
 3. The oligonucleotide of claim 1, wherein at least one of saidnucleotides has a modified nucleobase.
 4. The oligonucleotide of claim1, wherein at least one of said nucleotides has a modified sugar moiety.5. The oligonucleotide of claim 1, wherein at least one of said covalentlinkages of said oligonucleotide is a modified covalent linkage and atleast one of said nucleotides has a modified sugar moiety.
 6. Theoligonucleotide of claim 1 having at least two non-contiguousnucleotides having modified sugar moieties.
 7. The oligonucleotide ofclaim 1 having at least two non-contiguous nucleotides having modifiedsugar moieties, wherein at least one of said covalent linkages of saidoligonucleotide is a modified covalent linkage and at least one of saidnucleotides has a modified sugar moiety.
 8. The oligonucleotide of claim1 further comprising at least one lipophilic moiety which enhances thecellular uptake of said oligonucleotide.
 9. An oligonucleotidecomprising from 8 to 30 nucleotides connected by covalent linkages,wherein said oligonucleotide has a sequence specifically hybridizablewith a nucleic acid encoding a first isoform of a JNK protein, and saidsequence of said oligonucleotide is not specifically hybridizable with anucleic acid encoding a second isoform of said JNK protein, and whereinsaid oligonucleotide modulates the expression of said first isoform ofsaid JNK protein but not that of said second isoform of said JNKprotein.
 10. A pharmaceutical composition comprising the oligonucleotideof claim 1, or a bioequivalent thereof, and a pharmaceuticallyacceptable carrier.
 11. The pharmaceutical composition of claim 10,further comprising one or more compounds from the list consisting of astabilizing agent, a penetration enhancer, a carrier compound and achemotherapeutic agent.
 12. A pharmaceutical composition comprising aplurality of the oligonucleotides of claim 1, or bioequivalents thereof,and a pharmaceutically acceptable carrier.
 13. A method of treating ananimal having, suspected of having or prone to having ahyperproliferative disease comprising administering to said animal aprophylactically or therapeutically effective amount of thepharmaceutical composition of claim
 10. 14. A method of modulating theexpression of a JNK protein in cells or tissues comprising contactingsaid cells or tissues with the oligonucleotide of claim
 1. 15. A methodof modulating cell cycle progression in cultured cells or the cells ofan animal comprising administering to said cells an effective amount ofthe oligonucleotide of claim
 1. 16. A method of modulating, in culturedcells or the cells of an animal, the phosphorylation of a proteinphosphorylated by a JNK protein, wherein said method comprisesadministering to said cells an effective amount of the oligonucleotideof claim
 1. 17. A method of modulating, in cultured cells or the cellsof an animal, the expression of a cellular protein that promotes one ormore metastatic events, wherein said method comprises administering tosaid cells an effective amount of the oligonucleotide of claim
 1. 18.The oligonucleotide of claim 1 wherein said JNK protein is that of amammal.
 19. The oligonucleotide of claim 3 wherein said modifiednucleobase is 5-methylcytosine.
 20. An oligonucleotide comprising from 8to 30 nucleotides connected by covalent linkages, wherein saidoligonucleotide has a sequence specifically hybridizable with two ormore nucleic acids encoding different isoforms of a JNK protein andwherein said oligonucleotide modulates the expression of said two ormore isoforms of said JNK protein.
 21. A method of inhibiting the growthof a tumor in an animal comprising administering to said animal aneffective amount of the pharmaceutical composition of claim
 10. 22. Amethod of inhibiting the growth of a tumor in an animal comprisingadministering to said animal an effective amount of the pharmaceuticalcomposition of claim
 11. 23. A method of inducing apoptosis in a cellcomprising contacting a cell with an antisense oligonucleotidecomprising from 8 to 30 nucleotides connected by covalent linkages,wherein said oligonucleotide has a sequence specifically hybridizablewith a nucleic acid encoding a JNK2 protein and decreases the expressionof said JNK2 protein, so that apoptosis is induced.
 24. A method oftreating a human having a disease or condition characterized by areduction in apoptosis comprising administering to a human aprophylactically or therapeutically effective amount of an antisenseoligonucleotide comprising from 8 to 30 nucleotides connected bycovalent linkages, wherein said oligonucleotide has a sequencespecifically hybridizable with a nucleic acid encoding a human JNK2protein and decreases the expression of said human JNK2 protein.
 25. Themethod of claim 23 wherein the antisense oligonucleotide has a sequencecomprising SEQ ID NO:
 31. 26. The method of claim 24 wherein saiddisease or condition is prostate cancer.
 27. The method of claim 21wherein said tumor is a prostate tumor.
 28. A method of treating ananimal having a disease or condition associated with a JNK proteincomprising administering to said animal a therapeutically orprophylactically effective amount of the compound of claim 1 so thatexpression of the JNK protein is inhibited.
 29. The method of claim 28wherein said disease or condition is inflammation.
 30. The method ofclaim 28 wherein said disease or condition is fibrosis or a fibroticdisease or condition.
 31. The method of claim 30 wherein said fibroticdisease or condition is fibrotic scarring, peritoneal adhesions, lungfibrosis or conjunctival scarring.
 32. The method of claim 28 whereinthe disease or condition is a hyperproliferative disease or condition.33. The method of claim 32 wherein the hyperproliferative disease orcondition is cancer.